Human Bone Marrow Progenitor Cells Research Articles

Induction of apoptosis by remoxipride metabolites in HL60 and CD34+/CD19− human bone marrow progenitor cells: potential relevance to remoxipride-induced aplastic anemia

The antipsychotic agent, remoxipride [(S)-(−)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2,6-dimethoxybenzamide] has been associated with acquired aplastic anemia. We have examined the ability of remoxipride, three pyrrolidine ring metabolites and five aromatic ring metabolites of the parent compound to induce apoptosis in HL60 cells and human bone marrow progenitor (HBMP) cells. Cells were treated for 0–24 h with each compound (0–200 μM). Apoptosis was assessed by fluorescence microscopy in Hoechst 33342- and propidium iodide stained cell samples. Results were confirmed by determination of internucleosomal DNA fragmentation using gel electrophoresis for HL60 cell samples and terminal deoxynucleotidyl transferase assay in HBMP cells. The catechol and hydroquinone metabolites, NCQ436 and NCQ344, induced apoptosis in HL60 and HBMP cells in a time- and concentration dependent manner, while the phenols, NCR181, FLA873, and FLA797, and the derivatives formed by oxidation of the pyrrolidine ring, FLA838, NCM001, and NCL118, had no effect. No necrosis was observed in cells treated with NCQ436 but NCQ344 had a biphasic effect in both cell types, inducing apoptosis at lower concentrations and necrosis at higher concentrations. These data show that the catechol and hydroquinone metabolites of remoxipride have direct toxic effects in HL60 and HBMP cells, leading to apoptosis, while the phenol metabolites were inactive. Similarly, benzene-derived catechol and hydroquinone, but not phenol, induce apoptosis in HBMP cells [Moran et al., Mol. Pharmacol., 50 (1996) 610–615]. We propose that remoxipride and benzene may induce aplastic anemia via production of similar reactive metabolites and that the ability of NCQ436 and NCQ344 to induce apoptosis in HBMP cells may contribute to the mechanism underlying acquired aplastic anemia that has been associated with remoxipride.

Selective destruction of leukaemic cells by photo-activation of 5-aminolaevulinic acid-induced protoporphyrin-IX.

The effect of 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT) on the viability and proliferation of leukaemia/lymphoma cells as well as normal human lymphocytes has been investigated by flow cytometry-propidium iodide assay (FC-PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and bromodeoxyuridine (BrdU) incorporation and on clonogenic activity of normal human bone marrow progenitor cells by clonogenic methods. ALA-PDT (1 mM 5-ALA, 4 h, 18 J cm-2) reduces the number and/or suppressed proliferation of leukaemic cells of promyelocytic (HL60), B-cell-derived (DAUDI) and T-cell-derived (JURKAT) cell lines by 2 logs and that of the HEL erythroleukaemia cells by 77%. The effect of ALA-PDT on quiescent human lymphocytes is small (85% viable cells after ALA-PDT). The proliferation of lymphocytes subjected to ALA-PDT and induced with phytohaemagglutinin (PHA) decreases by 75% as compared to the untreated control. For normal human bone marrow progenitors, 58% of colony-forming units-granulocytes-macrophages (CFU-GM) and 55% burst-forming units-erythrocytes (BFU-E) activities are preserved.

The role of protein glycosylation inhibitors in the prevention of metastasis and therapy of cancer.

Oligosaccharide moieties of cell-surface glycoproteins are thought to be involved in recognition events during cancer metastasis and invasion. Swainsonine, an inhibitor of the Golgi alpha-mannosidase II, has been shown to block pulmonary colonization by tumor cells and stimulate components of the immune system. Swainsonine also abrogates much of the toxicity of chemotherapeutic agents and stimulates bone marrow hematopoietic progenitor cells, suggesting additional therapeutic applications. We are currently characterizing the ability of swainsonine to modify cell growth in human and murine bone marrow progenitor cells. Furthermore, we are examining crucial steps in metastasis that depend upon cell surface molecules that play a role in cell-matrix interactions. Our work shows that tumor cell adhesion to collagen IV in vitro is rapidly stimulated by cis-polyunsaturated fatty acids and is dependent on protein kinase C activity. We are investigating the hypothesis that integrins are critical components of this adhesion and are examining potential signal transduction pathways that lead to the modulation of cell adhesion.

Characterization of CKβ8 and CKβ8-1: Two Alternatively Spliced Forms of Human β-Chemokine, Chemoattractants for Neutrophils, Monocytes, and Lymphocytes, and Potent Agonists at CC Chemokine Receptor 1

Two new members of human beta-chemokine cDNA were isolated based on structural and functional similarities to human leukotactin-1. One of these clones was identical to the previously isolated human beta-chemokine, CKbeta8, whereas the other is a splicing variant of CKbeta8, therefore named CKbeta8-1. CKbeta8 was short in 51 nucleotides (17 amino acids) compared with CKbeta8-1. The mature proteins of CKbeta8-1 and CKbeta8 consisted of 116 and 99 amino acids with calculated molecular weights of 12,500 and 10,950, respectively. Both CKbeta8-1 and CKbeta8 were potent agonists at CCR1. These chemokines chemoattracted neutrophils, monocytes, and lymphocytes. They also significantly suppressed colony formation by human bone marrow, granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors. To our knowledge, this is the first example that an alternative splicing produces two active beta-chemokines from a single gene.

Characterization of CKβ8 and CKβ8-1: Two Alternatively Spliced Forms of Human β-Chemokine, Chemoattractants for Neutrophils, Monocytes, and Lymphocytes, and Potent Agonists at CC Chemokine Receptor 1

AbstractTwo new members of human β-chemokine cDNA were isolated based on structural and functional similarities to human leukotactin-1. One of these clones was identical to the previously isolated human β-chemokine, CKβ8, whereas the other is a splicing variant of CKβ8, therefore named CKβ8-1. CKβ8 was short in 51 nucleotides (17 amino acids) compared with CKβ8-1. The mature proteins of CKβ8-1 and CKβ8 consisted of 116 and 99 amino acids with calculated molecular weights of 12,500 and 10,950, respectively. Both CKβ8-1 and CKβ8 were potent agonists at CCR1. These chemokines chemoattracted neutrophils, monocytes, and lymphocytes. They also significantly suppressed colony formation by human bone marrow, granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors. To our knowledge, this is the first example that an alternative splicing produces two active β-chemokines from a single gene.

Development of a New Anti-CD4 Monoclonal Antibody (YG23) Which Inhibits the Formation of Colonies of Human Bone Marrow Progenitor Cells

Development of a New Anti-CD4 Monoclonal Antibody (YG23) Which Inhibits the Formation of Colonies of Human Bone Marrow Progenitor Cells

Neither Human Immunodeficiency Virus-1 (HIV-1) nor HIV-2 Infects Most-Primitive Human Hematopoietic Stem Cells as Assessed in Long-Term Bone Marrow Cultures

AbstractAttempts to clarify the pathophysiology of human immunodeficiency virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to the viral infection. To specifically address this question, we exposed highly purified subpopulations of human BM progenitor cells to various HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and expression in more-committed (CD34+CD38+) as well as most-primitive (CD34+CD38−) cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects of exposing hematopoietic stem cells to HIV. Our results show that HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in vitro with any significant frequency and infected cells are not present within LTCICs. Cytofluorometric analysis of CD34+ cells for surface molecules that facilitate HIV entry was consistent with the functional assay in that expression of virus receptors was predominantly on the more-committed subsets of BM progenitors. The failure to detect productive or latent HIV in the most-primitive human BM progenitor and stem cells has important implications for future therapeutic strategies, including those dealing with transduction of these cells with protective genes as a treatment modality for AIDS.

Neither Human Immunodeficiency Virus-1 (HIV-1) nor HIV-2 Infects Most-Primitive Human Hematopoietic Stem Cells as Assessed in Long-Term Bone Marrow Cultures

Attempts to clarify the pathophysiology of human immunodeficiency virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to the viral infection. To specifically address this question, we exposed highly purified subpopulations of human BM progenitor cells to various HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and expression in more-committed (CD34+CD38+) as well as most-primitive (CD34+CD38−) cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects of exposing hematopoietic stem cells to HIV. Our results show that HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in vitro with any significant frequency and infected cells are not present within LTCICs. Cytofluorometric analysis of CD34+ cells for surface molecules that facilitate HIV entry was consistent with the functional assay in that expression of virus receptors was predominantly on the more-committed subsets of BM progenitors. The failure to detect productive or latent HIV in the most-primitive human BM progenitor and stem cells has important implications for future therapeutic strategies, including those dealing with transduction of these cells with protective genes as a treatment modality for AIDS.

Changes in bone marrow inflammatory cell progenitors after inhaled allergen in asthmatic subjects.

Increases in inflammatory cell progenitors, particularly eosinophil/basophil colony-forming cells (Eo/B-CFU), occur in peripheral blood after allergen provocation. The role of bone marrow (BM) in these reactions is unclear. We examined the effect of allergen challenge on human bone marrow progenitor cell growth. Fifteen asthmatic subjects, eight dual responders (DR) and seven isolated early responders (IER), were challenged with inhaled allergen. BM aspirates were taken before and 24 h after challenge and progenitors were enumerated by a colony-forming assay. Eo/B-CFU numbers increased in both groups after allergen challenge (p < 0.0001). For DR, the increases were significant for BM incubated with optimal GMCSF and IL-5, but not with IL-3. For IER, the increases were significant for all three cytokines tested. At a suboptimal concentration of IL-5, there was a significant increase in the number of Eo/B-CFU after allergen in the DR, from 5.25 +/- 1.2 to 9.68 +/- 2.1 per 2.5 x 10(5) cells plated (p < 0.01), which was not demonstrated in the IER (p = 0.94). The responses at this concentration of IL-5 were different between groups (p < 0.05). These results demonstrate that inhaled allergen increases BM Eo/B-CFU, and that the bone marrow of dual responders is more responsive to IL-5 after allergen.

Variation in cell yield and proliferative activity of positive selected human CD34+ bone marrow cells along the circadian time scale.

Variations in cell yield and proliferative activity of human bone marrow (BM) progenitor cells were determined with flow cytometry along the 24-h (circadian) time scale. Equal volumes of BM were aspirated every 5 h, altogether 5 times in 5 healthy men. An average 6-fold higher yield of positive selected CD34+ cells occurred in each subject when BM was aspirated during the daytime and late afternoon, while a lower yield occurred during the night. Using all CD34+ cell yield data normalized to percentage of mean, a significant time-effect was found by ANOVA (p = 0.02) and a significant circadian rhythm was detected by the least-squares fit of a 24 h cosine (p = 0.02). The 95% confidence limits of the acrophase (time of highest values) were computed to be at midday between 10:24 and 14:48 h. A highly significant correlation (p = 0.001) was found between proliferation of positive selected CD34+ cells and the more mature myeloid precursor cells from the same BM aspirates, suggesting a common temporal pattern along the circadian time scale. However, no correlation was demonstrated between proliferation and cell yield of CD34+ selected cells, suggesting that mechanisms other than variation in proliferation may cause the circadian rhythm in stem cell yield. These circadian variations in stem cell yield and proliferation suggest that proper timing within 24 h may potentially be important regarding outcome from progenitor cell harvesting and treatment with haematopoietic growth factors.

Ability of Early Acting Cytokines to Directly Promote Survival and Suppress Apoptosis of Human Primitive CD34+CD38− Bone Marrow Cells With Multilineage Potential at the Single-Cell Level: Key Role of Thrombopoietin

AbstractPurified primitive progenitor/stem cells from bone marrow represent likely target populations for ex vivo expansion of stem cells to be used in high-dose chemotherapy or gene therapy. Whereas such primitive progenitor cells require combined stimulation by multiple cytokines for growth, some cytokines selectively promote viability rather than growth when acting individually. We investigated here for the first time the direct effects of cytokines on survival of primitive CD34+CD38− human bone marrow progenitor cells at the single-cell level. Interleukin-3 (IL-3) and the ligands for c-kit (KL) and flt3 (FL) had direct and selective viability-promoting effects on a small fraction of CD34+CD38− but not CD34+CD38+ progenitor cells. Interestingly, the recently cloned thrombopoietin (Tpo), although stimulating little growth, kept most CD34+CD38− progenitors viable after prolonged culture, maintaining twofold and fourfold more progenitors viable than KL and IL-3, respectively. A high fraction of these progenitors had a combined myeloid and erythroid differentiation potential, as well as capacity for prolonged production of progenitor cells under stroma-independent conditions. In addition, Tpo promoted viability of CD34+CD38− long-term culture-initiating cells, further supporting the idea that Tpo promotes viability of primitive human progenitor cells. Finally, Tpo suppressed apoptosis of CD34+CD38− cells in culture. Thus, the present studies show a novel effect of Tpo, implicating a potential role of this cytokine in maintaining quiescent primitive human progenitor cells viable.

Ability of Early Acting Cytokines to Directly Promote Survival and Suppress Apoptosis of Human Primitive CD34+CD38− Bone Marrow Cells With Multilineage Potential at the Single-Cell Level: Key Role of Thrombopoietin

Purified primitive progenitor/stem cells from bone marrow represent likely target populations for ex vivo expansion of stem cells to be used in high-dose chemotherapy or gene therapy. Whereas such primitive progenitor cells require combined stimulation by multiple cytokines for growth, some cytokines selectively promote viability rather than growth when acting individually. We investigated here for the first time the direct effects of cytokines on survival of primitive CD34+CD38- human bone marrow progenitor cells at the single-cell level. Interleukin-3 (IL-3) and the ligands for c-kit (KL) and flt3 (FL) had direct and selective viability-promoting effects on a small fraction of CD34+CD38- but not CD34+CD38+ progenitor cells. Interestingly, the recently cloned thrombopoietin (Tpo), although stimulating little growth, kept most CD34+CD38- progenitors viable after prolonged culture, maintaining twofold and fourfold more progenitors viable than KL and IL-3, respectively. A high fraction of these progenitors had a combined myeloid and erythroid differentiation potential, as well as capacity for prolonged production of progenitor cells under stroma-independent conditions. In addition, Tpo promoted viability of CD34+CD38- long-term culture-initiating cells, further supporting the idea that Tpo promotes viability of primitive human progenitor cells. Finally, Tpo suppressed apoptosis of CD34+CD38- cells in culture. Thus, the present studies show a novel effect of Tpo, implicating a potential role of this cytokine in maintaining quiescent primitive human progenitor cells viable.

Photodynamic effects of meso-tetra(4-sulfonatophenyl) porphine on human leukemia cells HEL andHL6, human lymphocytes and bone marrow progenitor cells

Meso-tetra(4-sulfonatophenyl)porphine (TPPS 4), in combination with a light dose of 14 J cm −2, has a profound negative effect on the proliferation and viability of leukemia cells HL60 (human promyelocytic leukemia) and HEL (human erythroleukemia), the viability of normal lymphocytes and the colony-forming activity of human bone marrow progenitor cells. However, normal leukocytes (monocytes, granulocytes) are, to a large extent, resistant to photodynamic treatment (PDT). Whilst DNA fragmentation suggesting apoptosis is induced in HL60 cells, accumulation in the interphase of the cell cycle (G 0/G 1, G 2/M) is mainly operative in the TPPS 4-mediated PDT of HEL cells. The “dark” effect of TPPS 4 on the cell viability is below 15% up to a concentration of 40 μM.

Thrombopoietin directly and potently stimulates multilineage growth and progenitor cell expansion from primitive (CD34+ CD38-) human bone marrow progenitor cells: distinct and key interactions with the ligands for c-kit and flt3, and inhibitory effects of TGF-beta and TNF-alpha.

Thrombopoietin (Tpo) is a primary regulator of megakaryocyte and platelet production. However, studies in c-mpl-deficient mice suggest that Tpo might also play an important role in early hemopoiesis. Here, the direct ability of Tpo to stimulate stroma-independent growth, multilineage differentiation, and progenitor cell expansion from single primitive CD34+ CD38- human bone marrow cells was investigated. Tpo alone stimulated limited clonal growth, but synergized with c-kit ligand (KL), flt3 ligand (FL), or IL-3 to potently enhance clonogenic growth. Whereas KL and FL in combination stimulated the clonal growth of only 3% of CD34+ CD38- cells, 40% of CD34+ CD38- cells were recruited by KL+FL+Tpo, demonstrating that Tpo promotes the growth of a high fraction of CD34+ CD38- progenitor cells. Additional cytokines (IL-3, IL-6, and erythropoietin (Epo)) did not significantly enhance clonal growth above that observed in response to KL+FL+Tpo. In contrast, Tpo enhanced clonogenic growth in response to KL+FL+IL-3+IL-6+Epo by as much as 80%, implicating a key role for this cytokine in early hemopoiesis. Importantly, we also demonstrate that the majority of Tpo-recruited CD34+ CD38- progenitor cells have a multilineage differentiation potential, and that Tpo promotes prolonged expansion of multipotent progenitors. Specifically, whereas progenitor cells were reduced in cultures containing only KL+FL, addition of Tpo resulted in 40-fold expansion of multipotent progenitors following a 14-day incubation. Finally, we identified inhibitors of Tpo-induced progenitor cell growth, in that TGF-beta as well as TNF-alpha almost completely abrogated the growth of CD34+ CD38- progenitor cells in response to Tpo alone as well as KL+FL+Tpo.

Comparison of Cytotoxicity of Mononucleoside Phosphotriester Derivatives Bearing Biolabile Phosphate Protecting Groups in Normal Human Bone Marrow Progenitor Cells

The effects of three mononucleoside phosphotriester derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT) which incorporate biolabile phosphate protecting groups, namely S-acetyl-2-thioethyl (MeSATE), S-(2-hydroxyethylsulfidyl)-2-thioethyl (DTE), and pivaloyloxymethyl (POM) were studied and compared to their nucleoside parent in human myeloid colony-forming cells. Moreover, the relative antiviral potency of these three pronucleotides were determined in primary human peripheral blood mononuclear cells infected with human immunodeficiency virus type 1. The results indicate that the SATE and DTE pro-moieties, as well as their degradation products, do not induce additional toxicity. The bis(MeSATE) phosphotriester derivative of AZT emerged as the most selective inhibitor with an in-vitro therapeutic index of the same order of magnitude as observed for AZT. This study has been extended to the corresponding bis(MeSATE) and bis(DTE) phosphotriester derivatives of 2′,3′-dideoxyuridine (ddU).

Comparison of benzimidazole nucleosides and ganciclovir on the in vitro proliferation and colony formation of human bone marrow progenitor cells

Recently we have shown that certain benzimidazole ribonucleosides are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. Because antiviral drugs used to treat HCMV and human immunodeficiency virus (HIV) infections can suppress marrow progenitors, we have evaluated the most promising of the new benzimidazoles for their effects on human bone marrow cells in vitro. In an initial study of the bone marrow toxicity of one of the most active compounds, 100 microM 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)-benzimidazole (BDCRB) inhibited cell proliferation by 20% over a 10 d period compared to 52% inhibition by 100 microM ganciclovir, the drug currently most used to treat HCMV infections. The effects of these drugs and selected other benzimidazole nucleosides were evaluated more extensively in haemopoietic progenitor cell colony formation assays. Colony formation was determined at 2 weeks and scored as either burst forming units-erythroid (BFU-E), or colony forming units-granulocyte/macrophage (CFU-GM). At the highest concentration tested, 100 microM BDCRB only moderately affected BFU-E or CFU-GM formation (31% and 47% inhibition, respectively). This concentration is 10-fold higher than that required to produce a 10000-fold reduction in virus titre. Evaluation of the 2-chloro analog of BDCRB (TCRB) which is less potent against HCMV, its 5'-deoxy analog (5'-dTCRB) which is more potent, and the 2-unsubstituted compound (DRB) gave the following order of haemopoietic toxicity: DRB > TCRB > or = 5'-dTCRB > BDCRB. In contrast to the benzimidazoles, ganciclovir decreased colony formation by 84% for BFU-E and 86% for CFU-GM at 100 microM. These results establish that certain benzimidazole nucleosides are less toxic to haemopoietic progenitors than the preferred drug now being used clinically for HCMV infections. The results also establish that different structure-activity relationships exist for antiviral activity and progenitor cell toxicity, thereby suggesting that different mechanisms are involved in the two types of drug action.

Efficient Retroviral Transduction of Human Bone Marrow Progenitor and Long-Term Culture-Initiating Cells: Partial Reconstitution of Cells From Patients With X-Linked Chronic Granulomatous Disease by gp91-phox Expression

The primary immunodeficiencies are attractive candidates for the development of gene therapy approaches based on the transduction of hematopoietic cells. We have constructed a high-titer recombinant retrovirus for expression of gp91-phox, deficiencies of which cause the X-linked form of chronic granulomatous disease (X-CGD). We have used this vector to transduce human bone marrow, using either unfractionated mononuclear cells or purified CD34+ cells as targets and evaluated several infection protocols. Efficient gene transfer to progenitors and long-term culture-initiating cells (LTC-IC) was obtained for each target population. Importantly for potential clinical application, this could be achieved without the use of exogenous cytokines or polybrene. Progenitors representing each of the lineages detectable in vitro were transduced at equal efficiencies. The vector was shown partially to restore gp91-phox deficiency and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in transduced cells derived from X-CGD patients. These data demonstrate that it is possible to transduce primitive human hematopoietic cells efficiently and reconstitute NADPH oxidase.

Effect of recombinant human hemoglobin on human bone marrow progenitor cells: protection and reversal of 3′ -azido-3′ -deoxythymidine-induced toxicity

Long-term therapy of AIDS patients with 3′ -azido-3′ -deoxythymidine (AZT) is limited by hematopoietic toxicity. While the mechanism(s) of this toxicity remain elusive, various strategies are being developed to reduce these toxic effects including combination therapy with non-myelotoxic anti-human immunodeficiency virus (HIV) drugs and/or administration of protective or rescue agents, such as cytokines and growth factors. Using a physiologically relevant human CD34+ bone marrow cell liquid culture system, a crosslinked human recombinant hemoglobin (rHb), currently in Phase II clinical trials, was investigated for effects on hematopoiesis and for its potential in protecting or reversing AZT-induced hematopoietic toxicity. These investigations demonstrated that 0.01, 0.1, or 1 μM human rHb did not affect the proliferation of erythroid or myeloid lineage cells. A concentration of 1 μM rHb partially protected erythroid lineage cells from an inhibition of proliferation induced by 0.1 and 1 μM AZT. Inhibition of proliferation of cells previously exposed to AZT was not reversed at this concentration. These data suggest that human rHb may be of benefit in reducing the toxic effects of AZT in the bone marrow of AIDS patients.

The FLT3 Ligand Is a Direct and Potent Stimulator of the Growth of Primitive and Committed Human CD34+ Bone Marrow Progenitor Cells In Vitro

The present studies investigated the effects of the recently cloned flt3 ligand (FL) on the in vitro growth and differentiation of primitive and committed subsets of human CD34+ bone marrow (BM) progenitor cells. FL alone was a weak growth stimulator of CD34+ BM cells, but synergistically and directly enhanced colony formation in combination with interleukin (IL) 3, granulocyte colony-stimulating factor (G-CSF), CSF-1, granulocyte macrophage (GM) CSF stem cell factor (SCF), and IL-6. FL and SCF were equally effective in stimulating colony formation in combination with IL-3. However, the tri-factor combination of FL + IL-3 + SCF stimulated 2.3-fold and 2.5-fold more colonies than FL + IL-3 and SCF + IL-3, respectively. These additional recruited progenitors appeared to be predominantly located in a primitive (CD71-) subset of the CD34+ progenitors, as 4.5-fold more colonies were formed by CD34+CD71- cells in response to FL + IL-3 + SCF than to FL + IL-3 or SCF + IL-3. Similar findings were observed in serum-containing and serum-deprived cultures. Whereas FL did not enhance burst-forming unit-erythroid (BFU-E) colony formation of CD34+ BM cells in the presence of serum, a low number of BFU-E colonies were formed in response to FL plus erythropoietin (Epo) under serum-deprived conditions. In addition, FL both in serum-containing and serum-deprived cultures stimulated colony formation of more committed myeloid progenitors in CD34+CD71+ BM cells. Thus, FL potently stimulates the growth of primitive and more committed human BM progenitor cells.

Protection and rescue from 2',3'-dideoxypyrimidine nucleoside analog toxicity by hemin in human bone marrow progenitor cells.

Long-term therapy of AIDS patients with 3'-azido-3'-deoxythymidine (AZT) remains of concern because of resulting hematopoietic toxicity. While the mechanism(s) of this toxicity remains elusive, alternative strategies are being developed to reduce these toxic effects, including combination therapy with nonmyelotoxic antihuman immunodeficiency virus drugs and/or administration of protective or rescue agents, including cytokines and growth factors. By using a particularly relevant human CD34+ liquid culture system, the unique profiles of dideoxynucleoside (ddN) toxicities to both proliferation and differentiation were demonstrated, with decreased potencies in the order of 3'-fluoro-3'-deoxythymidine (FLT) = 3'-amino-3'-deoxythymidine (AMT) = 2',3'-dideoxycytidine > AZT for inhibition of proliferation and in the order of FLT = AMT > AZT >> 2',3'-dideoxycytidine for inhibition of hemoglobin synthesis. Hemin selectively protected erythroid-lineage human burst-forming unit-erythroid cells from AZT- and AMT-induced inhibition but had no effect on FLT toxicity under similar conditions. Myeloid-lineage human CFU-granulocyte-macrophages were also not protected by hemin against all three ddN analogs. The simultaneous exposure of cells to hemin and AZT resulted in a complete protection of both cell proliferation and hemoglobin synthesis. In contrast, in reversal studies only the inhibition of the percentage of hemoglobin-synthesizing cells returned to control levels, but the inhibition of proliferation of cells previously exposed to AZT was not reversed by hemin. These studies further define the unique and multifactorial mechanism(s) of ddN-induced toxic effects during hematopoietic development of pluripotent stem cells and suggest that the use of hemin could be beneficial in alleviating the toxicity of certain ddN analogs.