CD34 Progenitor Cells Research Articles

Deregulated expression of promyelocytic leukemia zinc finger protein in B-cell chronic lymphocytic leukemias does not affect cyclin A expression.

The promyelocytic leukemia zinc finger (PLZF) gene encodes a transcription factor expressed in myeloid, lymphoid and CD34(+) progenitor cells. Structurally related to BCL-6, which is involved in human lymphoma, PLZF may have a role in proliferation, differentiation and survival of hematopoietic cells, that could be mediated by transcriptional repression of the cyclin A gene. Quantitative competitive reverse transcription-polymerase chain reaction was used to measure the levels of expression of PLZF and cyclin A in normal leukocyte subsets (including CD19(+) lymphocytes, n=21) and malignant B lymphocytes (including B-chronic lymphocytic leukemias [B-CLL], n=63). Results obtained with this method were confirmed by Western and Northern blot analysis. Transactivation assays were performed using an expression construct for PLZF and two cyclin A promoter luciferase reporters in an Epstein-Barr virus (EBV)-transformed B-cell line. Cyclin A expression, cell growth kinetics, and cell cycle were analysed in stable clones of the Burkitt lymphoma (BL) B-cell line DG75 with inducible expression of PLZF, generated using the tetracycline-regulated expression system. Expression of PLZF was 100-fold downregulated in 90% B-CLL (56/63) compared to normal B lymphocytes (P<0.001). B-CLL patients with the highest levels of PLZF had a poorer survival (P<0.013). In transactivation assays, PLZF inhibited the activity of the cyclin A reporters by 50%, demonstrating that PLZF can repress cyclin A expression in non-malignant B lymphocytes. However, in B-CLL patients, the level of cyclin A expression was found to be within the normal range. Altered PLZF function in B lymphoid malignancies was further corroborated in the PLZF-regulatable DG75 clones, where induction of PLZF expression did not significantly alter the levels of cyclin A expression, the cell growth kinetics, or the cell cycle phase distribution. The lower survival of patients with the highest levels of PLZF suggests that this protein may be a marker of progression in B-CLL. The absence of co-ordinated regulation of PLZF and cyclin A genes in B-CLL and in a malignant B-cell line may indicate a loss of cyclin A control by PLZF in B-CLL and other B-cell disorders. Deregulation of PLZF could thus play a role in B-cell malignancy.

Enhanced endothelialization and microvessel formation in polyester grafts seeded with CD34+ bone marrow cells: Presented to the Western Vascular Society, Whistler, BC, Canada, September 11, 1998.

The authors have shown accelerated endothelialization on polyethylene terephthalate (PET) grafts preclotted with autologous bone marrow. Bone marrow cells have a subset of early progenitor cells that express the CD34 antigen on their surfaces. A recent in vitro study has shown that CD34(+) cells can differentiate into endothelial cells. The current study was designed to determine whether CD34(+) progenitor cells would enhance vascular graft healing in a canine model. The authors used composite grafts implanted in the dog's descending thoracic aorta (DTA) for 4 weeks. The 8-mm x 12-cm composite grafts had a 4-cm PET graft in the center and 4-cm standard ePTFE grafts at each end. The entire composite was coated with silicone rubber to make it impervious; thus, the PET segment was shielded from perigraft and pannus ingrowth. There were 5 study grafts and 5 control grafts. On the day before surgery, 120 mL bone marrow was aspirated, and CD34(+) cells were enriched using an immunomagnetic bead technique, yielding an average of 11.4 +/- 5. 3 x 10(6). During surgery, these cells were mixed with venous blood and seeded onto the PET segment of composite study grafts; the control grafts were treated with venous blood only. Hematoxylin and eosin, immunocytochemical, and AgNO(3 )staining demonstrated significant increases of surface endothelialization on the seeded grafts (92% +/- 3.4% vs 26.6% +/- 7.6%; P =.0001) with markedly increased microvessels in the neointima, graft wall, and external area compared with controls. In dogs, CD34(+) cell seeding enhances vascular graft endothelialization; this suggests practical therapeutic applications. (Blood. 2000;95:581-585)

Isolation, characterization, and chondrogenic potential of human bone marrow-derived multipotential stromal cells.

Multipotential bone marrow stromal cells have the ability to differentiate along multiple connective tissue lineages including cartilage. In this study, we developed an efficient and reproducible procedure for the isolation of stromal cells from bone marrow aspirates of normal human donors based on the expression of endoglin, a type III receptor of the transforming growth factor-beta (TGF-beta) receptor family. We demonstrate that these cells have the ability of multiple lineage differentiation. Stromal cells represented 2-3% of the total mononuclear cells of the marrow. The cells displayed a fibroblastic colony formation in monolayer culture and maintained similar morphology with passage. Expression of cell surface molecules by flow cytometry displayed a stable phenotype with culture expansion. When cocultured with hematopoietic CD34(+) progenitor cells, stromal cells were able to maintain their ability to support hematopoiesis in vitro. Culture expanded stromal cells were placed in a 3-dimensional matrix of alginate beads and cultured in serum-free media in the presence of TGFbeta-3 for chondrogenic lineage progression. Increased expression of type II collagen messenger RNA was observed in the TGFbeta3 treated cultures. Immunohistochemistry performed on sections of alginate beads detected the presence of type II collagen protein. This isolation procedure for stromal cells and the establishment of the alginate culture system for chondrogenic progression will contribute to the understanding of chondrogenesis and cartilage repair.

Pure red cell aplasia complicated by angioimmunoblastic T‐cell lymphoma: Humoral factor plays a main role in the inhibition of erythropoiesis from CD34 progenitor cells

Pure red cell aplasia complicated by angioimmunoblastic T‐cell lymphoma: Humoral factor plays a main role in the inhibition of erythropoiesis from CD34 progenitor cells

Gastrointestinal epithelium is an early extrathymic site for increased prevalence of CD34(+) progenitor cells in contrast to the thymus during primary simian immunodeficiency virus infection.

The objective of this study was to determine the effects of primary simian immunodeficiency virus (SIV) infection on the prevalence and phenotype of progenitor cells present in the gastrointestinal epithelia of SIV-infected rhesus macaques, a primate model for human immunodeficiency virus pathogenesis. The gastrointestinal epithelium was residence to progenitor cells expressing CD34 antigen, a subset of which also coexpressed Thy-1 and c-kit receptors, suggesting that the CD34(+) population in the intestine comprised a subpopulation of primitive precursors. Following experimental SIVmac251 infection, an early increase in the proportions of CD34(+) Thy-1(+) and CD34(+) c-kit+ progenitor cells was observed in the gastrointestinal epithelium. In contrast, the proportion of CD34(+) cells in the thymus declined during primary SIV infection, which was characterized by a decrease in the frequency of CD34(+) Thy-1(+) progenitor cells. A severe depletion in the frequency of CD4-committed CD34(+) progenitors was observed in the gastrointestinal epithelium 2 weeks after SIV infection which persisted even 4 weeks after infection. A coincident increase in the frequency of CD8- committed CD34(+) progenitor cells was observed during primary SIV infection. These results indicate that in contrast to the primary lymphoid organs such as the thymus, the gastrointestinal epithelium may be an early extrathymic site for the increased prevalence of both primitive and committed CD34(+) progenitor cells. The gastrointestinal epithelium may potentially play an important role in maintaining T-cell homeostasis in the intestinal mucosa during primary SIV infection.

Identification of human T-lymphoid progenitor cells in CD34+ CD38low and CD34+ CD38+ subsets of human cord blood and bone marrow cells using NOD-SCID fetal thymus organ cultures.

In contrast to myeloid and B-lymphoid differentiation, which take place in the marrow environment, development of T cells requires the presence of thymic stromal cells. We demonstrate in this study that human CD34+, CD34+ CD38+ and CD34+ CD38(low) cells from both cord blood and adult bone marrow reproducibly develop into CD4+ CD8+ T cells when introduced into NOD-SCID embryonic thymuses and further cultured in organotypic cultures. Such human/mouse FTOC fetal thymic organ culture) thus represents a reproducible and sensitive system to assess the T-cell potential of human primitive progenitor cells. The frequency of T-cell progenitors among cord-blood-derived CD34+ cells was estimated to be 1/500. Furthermore, the differentiation steps classically observed in human thymus were reproduced in NOD-SCID FTOC initiated with cord blood and human marrow CD34+ cells: immature human CD41(low) CD8- sCD3- TCR alphabeta- CD5+ CD1a+ T cells were mixed with CD4+ CD8+ cells and more mature CD4+ CD8- TCR alphabeta+ cells. However, in FTOC initiated with bone marrow T progenitors, <10% double-positive cells were observed, whereas this proportion increased to 50% when cord blood CD34+ cells were used, and most CD4+ cells were immature T cells. These differences may be explained by a lower frequency of T-cell progenitors in adult samples, but may also suggest differences in the thymic signals required by bone marrow versus cord blood T progenitors. Finally, since cytokine-stimulated CD34+ CD38(low) cells retained their ability to generate T cells, these FTOC assays will be of value to monitor, when combined with other biological assays, the influence of different expansion protocols on the potential of human stem cells.

CD34(+)/interleukin-5Ralpha messenger RNA+ cells in the bronchial mucosa in asthma: potential airway eosinophil progenitors.

Eosinophil differentiation is thought to occur by the action of interleukin (IL)-5 on CD34(+) progenitor cells. The allergen-induced increase in eosinophil numbers in isolated airway preparations in vitro, and detection of increased numbers of circulating CD34(+) cells in atopic subjects, led us to the hypothesis that the eosinophil infiltration of the airway in asthma may result from local mucosal differentiation, in addition to recruitment from the bone marrow. We examined CD34(+) cell numbers by immunohistochemistry and IL-5 receptor alpha (IL-5Ralpha) messenger RNA (mRNA) expression by in situ hybridization in bronchial biopsies from atopic asthmatic patients, and from atopic and nonatopic control subjects. CD34(+) cell numbers were increased in the airway in atopic asthmatic and atopic nonasthmatic subjects. In contrast, CD34(+)/ IL-5Ralpha mRNA+ cells were increased in asthmatic subjects when compared with both atopic and nonatopic control subjects. Airway numbers of CD34(+)/IL-5Ralpha mRNA+ cells were correlated to airway caliber in asthmatic subjects and to eosinophil numbers. These findings support the concept that eosinophils may differentiate locally in the airway in asthma.

T cells recognizing leukemic CD34(+) progenitor cells mediate the antileukemic effect of donor lymphocyte infusions for relapsed chronic myeloid leukemia after allogeneic stem cell transplantation.

Adoptive immunotherapy with donor lymphocyte infusions (DLI) is an effective treatment for relapsed chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. To identify the effector and target cell populations responsible for the elimination of the leukemic cells in vivo we developed an assay to measure the frequency of T lymphocyte precursor cells capable of suppressing leukemic progenitor cells. Target cells in this assay were CML cells that were cultured in the presence of stem cell factor, interleukin 3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. [3H]thymidine incorporation at day 7 represented the proliferation of the progeny of the CD34(+) CML progenitor cells, and not of the more mature CD34(-) CML cells. Effector cells were mononuclear cells, which were used in a limiting dilution analysis to measure the frequencies of CML progenitor cell-inhibitory lymphocyte precursors (PCILp) in peripheral blood of seven patients before and after DLI for relapsed CML. In the six patients who entered complete remission, a 5- to 100-fold increase of PCILp was found during the clinical response. In the patient with resistant relapse the frequency of PCILp was <10 per ml before and after DLI. Leukemia-reactive helper T lymphocyte precursor frequencies remained unchanged after DLI. A significant increase in cytotoxic T lymphocyte precursor frequency against more mature leukemic cells was found in only two responding patients. These results indicate that T cells specifically directed against CD34(+) CML progenitor cells mediate the antileukemic effect of DLI.

Anti-HIV-1 and chemotactic activities of human stromal cell-derived factor 1alpha (SDF-1alpha) and SDF-1beta are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage.

CD26 is a leukocyte-activation antigen that is expressed on T lymphocytes and macrophages and possesses dipeptidyl peptidase IV (DPPIV) activity, whose natural substrates have not been identified yet. CXC chemokines, stromal cell-derived factor 1alpha (SDF-1alpha) and 1beta (SDF-1beta), sharing the receptor CXCR-4, are highly efficacious chemoattractants for resting lymphocytes and CD34(+) progenitor cells, and they efficiently block the CXCR-4-mediated entry into cells of T cell line tropic strains of HIV type 1 (HIV-1). Here we show that both the chemotactic and antiviral activities of these chemokines are abrogated by DPPIV-mediated specific removal of the N-terminal dipeptide, not only when the chemokines are produced in transformed mouse L cell line to express human CD26 but also when they were exposed to a human T cell line (H9) physiologically expressing CD26. Mutagenesis of SDF-1alpha confirmed the critical requirement of the N-terminal dipeptide for its chemotactic and antiviral activities. These data suggest that CD26-mediated cleavage of SDF-1alpha and SDF-1beta likely occurs in human bodies and promotes HIV-1 replication and disease progression. They may also explain why memory function of CD4(+) cells is preferentially lost in HIV-1 infection. Furthermore, CD26 would modulate various other biological processes in which SDF-1alpha and SDF-1beta are involved.

Gene transfer of alpha interferon into hematopoietic stem cells

Gene transfer or gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. IFN-α has been used in the management of leukemia, and gene transfer of the IFN-α gene into hematopoietic progenitor cells may have great potential for the treatment of chronic myelogenous leukemia (CML). Therefore, we examined the ability of adenovirus (Ad)-IFN-α gene construct to transfect normal bone marrow hematopoietic CD34 + stem cells and the production of IFN-α protein by these cells. Ad-cytomegalovirus (CMV) promoter-driven IFN-α at multiple doses was assessed to transfect highly purified CD34 + cells in liquid culture. Optimal transduction of CD34 + cells with the AdCMV-IFN-α construct was achieved using 120 plaque forming units (pfu). Flow cytometric determinations revealed that there was no significant difference in CD34 + cell viability for the 8 or 12-h transfection periods. Immunoassay of IFN-α produced by CD34 + cells shows that IFN-α levels increased several fold in transfected cells and this was not seen in CD34 + cells transfected with the heme oxygenase gene (HO-1). These in vitro data suggest that adenovirus-mediated gene transfer of IFN-α into hematopoietic stem cells can be achieved and that the IFN-α protein is produced by viable CD34 progenitor cells.

Fas Antigen (CD95) Expression in Peripheral Blood Progenitor Cells from Patients with Leukaemia and Lymphoma

Fas antigen (CD95) is a cell surface receptor belonging to the tumour necrosis factor/nerve growth factor superfamily and is able to induce apoptosis when triggered by it's natural ligand or an anti-Fas antibody. Fas expression is low on CD34+ bone marrow (BM) progenitor cells, but is increased by various cytokines in vitro. We investigated Fas expression on CD34+ cells from 39 peripheral blood progenitor cell (PBPC) harvests and from 5 normal BM harvests by dual colour flow cytometry to determine if Fas expression was altered during mobilisation. By including calibrated microbeads during flow cytometry, we quantified the number of Fas antigen molecules per cell. A low percentage of PBPC (22%) and normal BM (23%) CD34+ cells expressed Fas antigen. Fas expression varied on CD34+ cells from different diseases and the highest expression was found in ALL (52%). There was a significant three fold increase in the number of Fas molecules/cell expressed on CD34+ cells (PBPC 6,230 molecules/cell, BM 2,236; p0.0003). This level of expression was considerably less than that for CD3/CD19 lymphocytes (33,095 molecules/cell) and CD 14 monocytes (47,467 molecules/cell) in the PBPC harvest. In conclusion, mobilisation including the use of growth of factors, has minimal effect on CD34 progenitor cell Fas expression.

Human immunodeficiency virus infection impairs hemopoiesis in long-term bone marrow cultures: nonreversal by nucleoside analogues.

Hematologic abnormalities are often seen in patients infected with human immunodeficiency virus (HIV). The effect of HIV infection of bone marrow stroma on support of uninfected CD34 progenitor cells in long-term bone marrow culture (LTBMC) was investigated. Results show that HIV-infected bone marrow stroma was unable to adequately support CD34 progenitor cells in vitro. Zidovudine or didanosine was added to cultures in an attempt to reverse the suppressive effects exerted by HIV and to determine whether such suppression was mediated by transfer of HIV infection to progenitor cells. Didanosine failed to reduce the suppressive effects of HIV, whereas zidovudine compounded the observed suppression. HIV infection of bone marrow stroma, while reducing the production of nonadherent cells, did not increase apoptosis and cell death in such cells. In contrast, zidovudine enhanced apoptosis and cell death in nonadherent cells produced by both HIV-infected and control LTBMC.

Bone Marrow Versus Peripheral Blood Progenitor Cells CD34 Selection in Patients With Non-Hodgkin's Lymphomas: Different Levels of Tumor Cell Reduction. Implications for Autografting

Human CD34+ selected cells are able to reconstitute hematopoiesis in patients receiving a myeloablative treatment. Although the role of reinfused tumor cells contaminating the grafts on the determination of postautograft relapses remains unclear, the major interest of CD34+ cell selection is to reduce the tumor contamination of the graft. This can be achieved if tumor cells do not express the CD34 antigen. We previously showed that this approach was effective with bone marrow (BM) collections in patients with non-Hodgkin's lymphoma (NHL). Because peripheral blood progenitor cells (PBPC) allow faster hematologic recovery than BM and are expected to contain less tumor contamination, we have compared the results of CD34+ cell selection in 35 BM and 16 PBPC from 48 patients with NHL. The PBPC were collected after a course of chemotherapy followed by granulocyte colony-stimulating factor (G-CSF ) administration. The data showed that the final CD34+ cell purity achieved with PBPC was higher than with BM (medians, 70% v 50%; P = .02). The CD34+ cell recovery was also better for PBPC (medians, 42% v 24%; P = .001). Tumor contamination was assessed by detection of BCL2/JH rearrangement using polymerase chain reaction (PCR) in 38 of 48 patients (22 BM, 16 PBPC). In addition, immunoglobulin heavy chain gene (IgH) rearrangements were investigated using PCR with consensus IgH primers. At harvesting, 10 of 22 BM and two of 16 PBPC contained BCL2/JH+ cells, one of 22 BM and 14 of 16 PBPC contained abnormal IgH+ cells (one PBPC contained both BCL2/JH+ and abnormal IgH+ cells) at harvesting. However, because lymphoma tissue specimens from patients at diagnosis were not available, the malignant character of IgH rearrangements could not be confirmed by sequencing and probing with allele-specific nucleotides. After CD34+ cell selection, a reduction to below the level of detection of BCL2/JH+ cells of BM and PBPC was effective in seven of 12 informative selections. In contrast, a reduction to below the level of detection of abnormal IgH+ cells was effective in only three of 15 informative selections. However, the detection of cells with an abnormal IgH pattern in the context of chemotherapy plus G-CSF progenitor mobilization in patients with NHL and its correlation with actual tumor contamination needs further investigation.

Bone Marrow Versus Peripheral Blood Progenitor Cells CD34 Selection in Patients With Non-Hodgkin's Lymphomas: Different Levels of Tumor Cell Reduction. Implications for Autografting

AbstractHuman CD34+ selected cells are able to reconstitute hematopoiesis in patients receiving a myeloablative treatment. Although the role of reinfused tumor cells contaminating the grafts on the determination of postautograft relapses remains unclear, the major interest of CD34+ cell selection is to reduce the tumor contamination of the graft. This can be achieved if tumor cells do not express the CD34 antigen. We previously showed that this approach was effective with bone marrow (BM) collections in patients with non-Hodgkin's lymphoma (NHL). Because peripheral blood progenitor cells (PBPC) allow faster hematologic recovery than BM and are expected to contain less tumor contamination, we have compared the results of CD34+ cell selection in 35 BM and 16 PBPC from 48 patients with NHL. The PBPC were collected after a course of chemotherapy followed by granulocyte colony-stimulating factor (G-CSF ) administration. The data showed that the final CD34+ cell purity achieved with PBPC was higher than with BM (medians, 70% v 50%; P = .02). The CD34+ cell recovery was also better for PBPC (medians, 42% v 24%; P = .001). Tumor contamination was assessed by detection of BCL2/JH rearrangement using polymerase chain reaction (PCR) in 38 of 48 patients (22 BM, 16 PBPC). In addition, immunoglobulin heavy chain gene (IgH) rearrangements were investigated using PCR with consensus IgH primers. At harvesting, 10 of 22 BM and two of 16 PBPC contained BCL2/JH+ cells, one of 22 BM and 14 of 16 PBPC contained abnormal IgH+ cells (one PBPC contained both BCL2/JH+ and abnormal IgH+ cells) at harvesting. However, because lymphoma tissue specimens from patients at diagnosis were not available, the malignant character of IgH rearrangements could not be confirmed by sequencing and probing with allele-specific nucleotides. After CD34+ cell selection, a reduction to below the level of detection of BCL2/JH+ cells of BM and PBPC was effective in seven of 12 informative selections. In contrast, a reduction to below the level of detection of abnormal IgH+ cells was effective in only three of 15 informative selections. However, the detection of cells with an abnormal IgH pattern in the context of chemotherapy plus G-CSF progenitor mobilization in patients with NHL and its correlation with actual tumor contamination needs further investigation.

Adenovirus mediated alpha interferon (IFN-alpha) gene transfer into CD34+ cells and CML mononuclear cells.

Gene transfer or gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. Interferon-alpha (IFN-alpha) has been used in the management of leukemia but its diverse adverse activities with multiple potential side effects, possibly unrelated to therapeutic targets, may negatively influence the ability of IFN-alpha to treat this disorder. Therefore, we examined the ability of adenovirus (Ad)-IFN-alpha gene construct to transfect normal (CD34+ cells) and chronic myelogenous leukemia (CML) bone marrow mononuclear cells (BMMNC) and the transient overexpression of IFN-alpha in these cells. Ad-cytomegalovirus promoter driven IFN-alpha (AdCMV-IFN-alpha) at multiple doses was assessed to transfect highly purified CD34+ cells in liquid culture, and optimal transduction of CD34+ cells was achieved using 120 plaque forming units. Flow cytometric determinations revealed that there was no significant difference in cell viability for the 4 h or 24 h transfection periods. Immunoassay of IFN-alpha produced by CD34+ cells shows that IFN-alpha levels increased several fold in transfected cells. Transient expression of the IFN-alpha gene did not suppress proliferation of CD34+ progenitors as indicated by BFU-E or colony forming units-granulocyte-macrophage (CFU-GM) growth. Reverse transcriptase/polymerase chain reaction analysis of RNA from CD34+ harvested CFU-GM progenitor cells demonstrated transient IFN-alpha mRNA expression. Similarly, CML BMMNC were transfected with AdCMV-IFN-alpha under similar conditions as described for CD34+ cells. BMMNC cells exposed to adenovirus for 24 h and 48 h were found to express IFN-alpha at a substantial level. This in vitro data suggest that Ad-mediated gene transfer of IFN-alpha into hematopoietic stem cells can be achieved and that the IFN-alpha gene can be translated into its specific mRNA in CD34 progenitor cells.

Thrombopoietin-independent effect of interferon-gamma on the proliferation of human megakaryocyte progenitors.

Flow cytometric study revealed that almost all CD34+ cells in human umbilical cord blood expressed interferon-gamma receptor (IFN-gammaR). To clarify the precise functional roles of IFN-gammaR in human CD34+ cells, we examined the effect of IFN-gamma alone and in combination with various cytokines on the growth of haemopoietic progenitor cells in CD34+ cells using a serum-free clonal culture. Surprisingly, IFN-gamma alone supported only megakaryocyte (MK) colonies in a dose-dependent manner with a plateau level at 1000 U/ml of IFN-gamma. IFN-gamma at 1000 U/ml induced 10 +/- 1.2 MK colonies from 1 x 10(3) CD34+ cells, whereas thrombopoietin (TPO), interleukin (IL)-3, stem cell factor (SCF) or IL-6 alone induced 22 +/- 4.0, 22 +/- 4.2, 4 +/- 0.6 and 0 MK colonies, respectively. The addition of anti-IFN-gamma monoclonal antibody (mAb) to the IFN-gamma culture completely abrogated MK colony formation, whereas the mAb had no effect on TPO-dependent production of MK colonies. In contrast, although anti-TPO polyclonal Ab almost completely blocked TPO-dependent MK colony formation, it failed to inhibit the generation of MK colonies induced by IFN-gamma, suggesting that the observed effect of IFN-gamma on the proliferation of human MK progenitor cells is independent of TPO. The addition of IFN-gamma to culture with TPO or SCF significantly augmented the development of MK colonies, whereas it did not affect IL-3-dependent MK colony formation. Additionally, IFN-gamma induced the increase of DNA content of cultured glycoprotein IIb/IIIa-positive megakaryocytes. These results suggest that IFN-gamma may have regulatory roles in human megakaryocytopoiesis.

CD34 progenitor cell subset analyses in normal human bone marrow and marrow harvested after intermediate-dose chemotherapy.

Previous attempts to characterize harvested marrow and peripheral blood stem cell (PBSC) in order to predict time to and quality of engraftment post autologous bone marrow transplant (autoBMT) have included use of in vitro colony forming unit (CFU) assays. These assays are hampered by interlaboratory variability and are not uniformly predictive. CD34 quantification by flow cytometric technique has also been used to assess the quality of harvested marrow and PBSC. However, a lack of standardization has hampered direct comparison of published reports. We sought to characterize these early lineage-committed CD34+ progenitor cells from non-ficolled harvested marrow with six progenitor cell (PC) panels containing CD34 antibody plus two additional early lineage markers, using multiparameter flow cytometry. The specific gating technique including simultaneous CD34-PE vs. side scatter and forward vs. side scatter, was verified using morphologic analyses of sorted CD34+ cells. An ungated file was initially acquired to assess total CD34+ content. A second file using a CD34 threshold was then acquired to resolve lineage-committed subsets. The % CD34+ cells as well as cells/microliter of bone marrow was calculated using cell counts at the time of marrow harvest. Bone marrow (mean total cell dose = 3.8 x 10(5)/kg), obtained from 42 normal donors for allogeneic transplantation was first analyzed. CD34+ cells comprised a mean 1.3% of non-ficolled marrow, with 328 CD34+ cells/microliter, and mean CD34+ cells collected was 4.8 x 10(6)/kg. While no significant differences in total cells harvested nor proportion of CD34+ cells was found, a significant decrease in CD34 cells/microliter (= 233, P = .0012) was found in cancer patients. The percentage of CD19+ and CD38+ progenitor cells was significantly increased, while CD5+ and CD71+ cells were decreased. The proportions of all other early lineage-committed CD34 subsets were not different. Measurement of lineage-committed CD34 progenitor cells is a useful technique to characterize harvested marrow and PBSC, and may be applied to predict time and quality of engraftment post ablative conditioning regimens.

Direct and reversible inhibition of platelet factor 4 on megakaryocyte development from CD34+ cord blood cells: comparative studies with transforming growth factor β1

Mechanisms of the action of platelet factor 4 (PF4) on the growth of megakaryocyte (MK) progenitor cells in CD34+ cord blood (CB) cells were studied in comparison with transforming growth factor beta1 (TGFbeta1). Development of MK from CD34+ CB cells in both plasma clot culture and liquid culture was significantly inhibited by either purified human PF4 and by recombinant human TGFbeta1. Inhibition of MK colony formation by PF4 was reversible because CD34+ cells preincubated with PF4 could regenerate colonies after washing and replating into secondary cultures. In contrast, TGFbeta1-preincubated CD34+ cells gave rise to few colonies following replating. Moreover, incubation of CD34+ cells with PF4 in liquid culture caused the increased number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells. In addition, PF4-preincubated CD34+ cells exhibited a higher potential in MK colony formation in the presence of 5-fluorouracil (5FU). These results demonstrate that both PF4 and TGFbeta1 inhibit MK development from CD34+ CB cells by different mechanisms, and suggest that PF4, unlike TGFbeta1, exerts its inhibitory effect on the growth of the target cells in a reversible manner which results in a preservation of a more immature and 5FU-resistant cell population.

Myeloid progenitor cell regulatory effects of vascular endothelial cell growth factor.

Vascular endothelial cell growth factor (VEGF) is a ligand for the tyrosine kinase receptor Flk-1/KDR and Flt1 and is considered to be an endothelial cell specific mitogen that plays an important role in angiogenesis. Since Flk-1 mRNA has been detected in primitive and more mature hematopoietic cells, recombinant human VEGF was evaluated for its influence on hematopoiesis, which was assayed as in vitro colony formation by myeloid progenitor cells from human bone marrow. VEGF enhanced colony formation by mature subsets of granulocyte-macrophage and erythroid progenitor cells that had been stimulated with a colony stimulating factor. In contrast, VEGF inhibited colony formation by more immature subsets of granulocyte-macrophage, erythroid and multipotential progenitor cells synergistically stimulated to proliferate with a colony stimulating factor and either steel factor or the ligand for the Flt-3 receptor tyrosine kinase. VEGF produced effects similar to those given above on purified CD34 progenitor cells from bone marrow and VEGF effects were neutralized by VEGF antibodies. However, when assessed for effects on single sorted CD34 cells, VEGF only enhanced or suppressed colony formation by granulocyte-macrophage progenitor cells and the amplitude of the response was less than that observed when populations of these cells were tested. In the single cell assays, VEGF had no effect on colony formation by erythroid or multipotential progenitors. These results suggest that the effects of VEGF, which were not species specific, are mediated by both direct and indirect actions on the progenitors and thereby identify new activities for this important factor.