Burst-forming Unit-erythroid Colony Formation Research Articles

Low-intensity pulsed ultrasound-mediated stimulation of hematopoietic stem/progenitor cell viability, proliferation and differentiation in vitro

Low-intensity pulsed ultrasound (LIPUS) stimulated the viability, proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) from fresh and cryopreserved peripheral blood leukapheresis product, as well as cord blood when applied for 10 min each day for 4 days. Cell viability, proliferation and differentiation were assessed on day 5 by viable cell counting, MTS proliferation assay, flow cytometry, and colony-forming unit assay. LIPUS stimulation: (i) enhanced the proliferation of fresh HSPC and maintained the viability of cryopreserved HSPC in vitro; (ii) did not affect the percentage of CD34(+) and CD14(+) cells; and (iii) enhanced burst-forming unit-erythroid colony formation. Hence, we suggest that this novel LIPUS stimulation approach might enhance the efficacy of clinical transplantation and cellular therapies using HSPC.

Myeloid-Derived Suppressor Cells (MDSC) Are Effectors of Bone Marrow Suppression in Lower Risk Myelodysplastic Syndromes (MDS).

Abstract 597The acquisition of genetic abnormalities that lead to ineffective hematopoiesis is a characteristic of MDS. This event is mediated in part by an interaction of inflammatory intermediates with the bone marrow (BM) microenvironment; however, the mechanism by which chromosomal damage occurs to the stem/progenitor compartment (HSC/HPC) is unknown. We have identified a unique population of myeloid derived suppressor cells (MDSC) bearing a LIN-HLA-DR-CD33+ surface phenotype. These MDSC elicit hematopoietic suppression through the elaboration of nitric oxide, arginase, and inflammatory cytokines. This study found that MDSCs accumulated in excess in the BM of patients with lower risk MDS (mean, 32.32% ± 13.3; n=12) compared to BM from healthy age matched controls (mean 2.1%±0.9; n=8) and non-MDS cancer patients (n=8 mean 2.06%±1.5 p<0.0001). Fluorescence in situ hybridization (FISH) was performed to determine whether MDSCs represent a distinct cell population from the abnormal MDS clone. MDSCs from BM of 5 patients having chromosomal abnormality were separated by FACS sorting based on LIN-HLA-DR-CD33+ phenotype and the presence of chromosomal abnormalities was determined in this population and compared to non-MDSCs. Monosomy 7 and deletion of 5q chromosomal abnormalities resided within the non-MDSC hematopoietic compartment. This indicates that MDSCs in low risk MDS patients may represent a unique cell population from the HPCs with clonal potential. Furthermore, the key cytokines involved in MDSC suppressive function, TGFβ, VEGF and IL-10, were higher in MDSCs isolated from MDS patients compared to controls. Using four-color immunostaining, we discovered that MDSCs are capable of direct cytotoxicity against autologous erythroid precursors (CD71+ and CD235a+), as evidenced by increased polarized granule mobilization toward the site of cellular contact. Moreover, it was observed that the accumulation of MDSC in the BM from MDS patients has an impact on hematopoietic differentiation. In the presence of MDSCs, the formation of BFU-E (burst forming unit erythroid) was significantly suppressed in all patients tested (n=6). In contrast depletion of the MDSC by FACS sorting reconstituted the formation of BFU-E (21± 4.2 in MDSC depleted bone marrow verse 0.8± 0.6 in unsorted BM cells, respectively). These data strongly suggest that the presence of MDSCs in the BM microenvironment of MDS patients contribute to suppression of HPC development. Based on these findings, a novel form of adaptive immunotherapy based on the induction of MDSC maturation can be envisioned. DAP12 (DNAX-activating protein of 12kDa) is an adaptor protein that mediates signaling of dentritic cell and monocyte maturation. In order to determine if DAP12 signaling induces MDSC maturation and reversal of suppressive function, a genetically modified, constitutively activated form was introduced into BM mononuclear cells (BM-MNC) using both recombinant adenoviral and lentiviral gene transduction. Infection of BM-MNC from MDS patients with constitutively active DAP12 increased expression of maturation surface markers CD14, CD15 and HLA-DR and increased BFU-E colony formation (31± 2.1 verse control 7±0.7) after 14 days. These results suggest that active signaling through DAP12 has potential therapeutic implications in MDS by driving maturation of immature myeloid cells reversing the inflammatory changes that contribute to malignant transformation. In conclusion, our previously unknown findings have begun to elucidate whether the presence of MDSCs in the BM microenvironment of MDS patients contributes to the pathogenesis of MDS by providing a suppressive microenvironment and repress BM maturation associated with impaired HSC/HPC development. Additionally, DAP12 may be a potential therapeutic pathway in which to stimulate MDSC maturation, reversing the suppressive effects on HSC/HPC development. Disclosures:No relevant conflicts of interest to declare.

Expression of P190 and P210 BCR/ABL1 in normal human CD34 + cells induces similar gene expression profiles and results in a STAT5-dependent expansion of the erythroid lineage

The P190 and P210 BCR/ABL1 fusion genes are mainly associated with different types of hematologic malignancies, but it is presently unclear whether they are functionally different following expression in primitive human hematopoietic cells. We investigated and systematically compared the effects of retroviral P190 BCR/ABL1 and P210 BCR/ABL1 expression on cell proliferation, differentiation, and global gene expression in human CD34(+) cells from cord blood. Expression of either P190 BCR/ABL1 or P210 BCR/ABL1 resulted in expansion of erythroid cells and stimulated erythropoietin-independent burst-forming unit-erythroid colony formation. By using a lentiviral anti-signal transducer and activator of transcription 5 (STAT5) short-hairpin RNA, we found that both P190 BCR/ABL1- and P210 BCR/ABL1-induced erythroid cell expansion were STAT5-dependent. Under in vitro conditions favoring B-cell differentiation, neither P190 nor P210 BCR/ABL1-expressing cells formed detectable levels of CD19-positive cells. Gene expression profiling revealed that P190 BCR/ABL1 and P210 BCR/ABL1 induced almost identical gene expression profiles. Our data suggest that the early cellular and transcriptional effects of P190 BCR/ABL1 and P210 BCR/ABL1 expression are very similar when they are expressed in the same human progenitor cell population, and that STAT5 is an important regulator of BCR/ABL1-induced erythroid cell expansion.

Adenovirus-mediated delivery of p53 results in substantial apoptosis to myeloma cells and is not cytotoxic to flow-sorted CD34(+) hematopoietic progenitor cells and normal lymphocytes.

Multiple myeloma (MM) is an incurable disease; therefore, there is a need for new modalities of treatment for this disease. We designed a study to test the sensitivity of MM cell lines, freshly isolated myeloma cells, and CD34(+) hematopoietic progenitor cells to adenovirus-mediated delivery of wild-type p53 (Ad-p53). Replication-deficient Ad-p53, previously used in phase I-II clinical trial for treatment of patients with solid tumors, was used in this study. Myeloma cells from seven MM cell lines with mutated or w.t. p53 and varying expression of bcl-2 were used. Fresh myeloma cells (CD38(bright)CD45(-)) and fresh CD34(+) hematopoietic stem cells and CD34(-) cells were purified by flow sorting of apheresis collections of MM patients undergoing high-dose chemotherapy and stem cell rescue. The effect of Ad-p53 on colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit erythroid (BFU-E) colony formation in methylcellulose was tested on purified CD34(+) and CD34(-) cells to evaluate bone marrow toxicity. Myeloma cells from cell lines, or freshly isolated myeloma cells, were sensitive to Ad-p53 only if they had mutated p53 and had low expression of bcl-2. CD34(+) cells were resistant to Ad-p53-mediated apoptosis, and CFU-GM and BFU-E colony formation was not affected by treatment with Ad-p53.Ad-p53 is a potent inducer of apoptosis in MM cell lines and in freshly isolated myeloma cells expressing low levels of bcl-2. Ad-p53 is not overtly cytotoxic to normal hematopoietic stem cells or normal lymphocytes; therefore, it could be considered for a phase I clinical trial of MM patients with mutated p53.

Globin Gene Silencing in Primary Erythroid Cultures: AN INHIBITORY ROLE FOR INTERLEUKIN-6

There are numerous similarities between the erythroid and megakaryocytic lineages which suggest that commitment to either lineage occurs relatively late in hematopoiesis. Commitment toward megakaryocyte development requires obligatory silencing of erythroid-specific genes. Therefore, we investigated the effects of interleukin-6, a known inducer of thrombocyte production, on globin gene expression during erythroid differentiation. Studies in K562 cells demonstrated inhibition of gamma globin gene mRNA production and chain biosynthesis in the presence of exogenous interleukin-6 which was abrogated by anti-interleukin-6 monoclonal antibody. Similar studies in primary erythroid progenitors showed inhibition of burst-forming unit-erythroid colony formation when interleukin-6 was added late in cultures with decreased gamma and beta globin gene mRNA production. Protein binding studies demonstrated an increase in activator protein-1 binding to its consensus sequence by 24 h of interleukin-6 treatment. Inhibition of activator protein-1 gene activity had no effect on gamma gene silencing by interleukin-6. A potential interleukin-6 response element was identified in the gamma globin gene. Interleukin-6 treatment led to a rapid increase in protein binding to the target DNA sequence. These results suggest that interleukin-6 may play an important role in globin gene silencing during megakaryocytic lineage commitment.

A Functional Analysis of Protooncogene Vav's Role in Adult Human Hematopoiesis

The Vav protooncogene is expressed almost exclusively in hematopoietic cells, but its role in regulating adult human hematopoietic cell development remains uncertain. To analyze Vav function in adult blood cell formation, we used antisense (AS) oligodeoxynucleotides (ODN) to disrupt its expression in normal and malignant human hematopoietic cells. Bone marrow or peripheral blood mononuclear cells (MNC) were obtained from consenting normal donors and patients with acute or chronic myelogenous leukemia (AML and CML, respectively) and polycythemia vera (PV). Adherent and T-cell-depleted (A-T-) MNC or CD34+ MNC were exposed to unmodified sense, antisense, or scrambled sequence ODN corresponding to codons 2-7 of Vav's mRNA sequence. Cells were then assayed for Vav mRNA expression by reverse transcription-polymerase chain reaction and Vav protein expression by Western binding. After showing that Vav-targeted AS ODN could specifically diminish Vav mRNA and protein expression, we assessed the ability of Vav-deficient cells to form myeloid and erythroid colonies in methyl-cellulose cultures. When normal CD34+ MNC were exposed to Vav AS ODN, no effect on colony-forming unit-granulocyte-macrophage (CFU-GM) or CFU-megakaryocyte colony formation was observed. In contrast erythroid colony growth was inhibited by a mean +/- SD of 62% +/- 16%. In patients with hematopoietic malignancies. Vav-targeted AS ODN inhibited CFU-GM colony formation in a sequence-specific and dose-dependent manner in 1 of 3 AML, 13 of 17 CML, and 2 of 2 PV patients. At the highest concentration used, the Vav AS ODN inhibited CFU-GM colony formation from 66% to 81% when compared with control cell colony growth. Burst-forming unit-erythroid (BFU-E) colony-formation was also assessed in 7 PV patients. The Vav-targeted AS ODN inhibited BFU-E colony formation in all by a mean +/- SD of 81% +/- 4%. These findings suggest that Vav function may not be easily complemented in a significant subset of normal adult erythroid progenitor cells and may also be necessary for myeloid progenitor cell growth in a variety of hematopoietic malignancies.

Transforming Growth Factor p (TGF-β), A Potent Inhibitor of Erythropoiesis: Neutralizing TGF-β Antibodies Show Erythropoietin as a Potent Stimulator of Murine Burst-Forming Unit Erythroid Colony Formation in the Absence of a Burst-Promoting Activity

Abstract Transforming growth factor beta (TGF-beta) is a bifunctional regulator of the growth of myeloid progenitors and is here demonstrated to directly inhibit the growth of primitive erythroid progenitors by 95% to 100% regardless of the cytokines stimulating growth. Autocrine TGF- beta production of primitive hematopoietic progenitors has previously been reported. In the present study, a neutralizing TGF-beta antibody (anti-TGF-beta) added to serum-containing cultures, resulted in a 3-, 4- , and 25-fold increase in burst-forming unit erythroid (BFU-E) colony formation in response to interleukin-4 (IL-4) plus erythropoietin (Epo), SCF plus Epo, and IL-11 plus Epo, respectively. The growth of BFU-E progenitors has been suggested to require a burst-promoting activity in addition to Epo. Accordingly, we observed no BFU-E colony formation in serum-containing cultures in response to Epo alone. In contrast, 50 BFU-E colonies were formed when anti-TGF-beta was included in the culture. In serum-free cultures, Epo also stimulated BFU-E colony formation in the absence of other cytokines, whereas anti-TGF- beta had no effect on the number of colonies formed. Quantitation of TGF-beta 1 in serum by an enzyme-linked immunosorbent assay method showed predominantly the presence of precursor (latent) TGF-beta 1, but also showed active TGF-beta 1 at a concentration sufficient to potently inhibit erythroid colony formation. Thus, neutralization of active TGF- beta 1 in serum shows that Epo alone is sufficient to stimulate the growth of murine BFU-E progenitors.

Transforming growth factor beta (TGF-beta), a potent inhibitor of erythropoiesis: neutralizing TGF-beta antibodies show erythropoietin as a potent stimulator of murine burst-forming unit erythroid colony formation in the absence of a burst-promoting activity

Transforming growth factor beta (TGF-beta) is a bifunctional regulator of the growth of myeloid progenitors and is here demonstrated to directly inhibit the growth of primitive erythroid progenitors by 95% to 100% regardless of the cytokines stimulating growth. Autocrine TGF-beta production of primitive hematopoietic progenitors has previously been reported. In the present study, a neutralizing TGF-beta antibody (anti-TGF-beta) added to serum-containing cultures, resulted in a 3-, 4-, and 25-fold increase in burst-forming unit erythroid (BFU-E) colony formation in response to interleukin-4 (IL-4) plus erythropoietin (Epo), SCF plus Epo, and IL-11 plus Epo, respectively. The growth of BFU-E progenitors has been suggested to require a burst-promoting activity in addition to Epo. Accordingly, we observed no BFU-E colony formation in serum-containing cultures in response to Epo alone. In contrast, 50 BFU-E colonies were formed when anti-TGF-beta was included in the culture. In serum-free cultures, Epo also stimulated BFU-E colony formation in the absence of other cytokines, whereas anti-TGF-beta had no effect on the number of colonies formed. Quantitation of TGF-beta 1 in serum by an enzyme-linked immunosorbent assay method showed predominantly the presence of precursor (latent) TGF-beta 1, but also showed active TGF-beta 1 at a concentration sufficient to potently inhibit erythroid colony formation. Thus, neutralization of active TGF-beta 1 in serum shows that Epo alone is sufficient to stimulate the growth of murine BFU-E progenitors.

Tumor Necrosis Factor (TNF)-α Directly Inhibits Human Erythropoiesis In Vitro: Role of p55 and p75 TNF Receptors

Two tumor necrosis factor receptors (TNFRs) with molecular weights of 55 kD (TNFR-p55) and 75 kD (TNFR-p75) have recently been identified and cloned. In previous studies, TNFR-p55 has been shown to exclusively mediate bidirectional effects of TNF-alpha on committed bone marrow granulocyte-macrophage progenitor cells, whereas both TNFR-p55 and TNFR-p75 can mediate inhibition of primitive progenitors requiring multiple cytokines to proliferate. We show here that TNF-alpha potently and directly inhibits the in vitro growth of committed erythroid progenitor cells in response to multiple cytokine combinations, and that TNF-alpha-induced inhibition of burst-forming unit-erythroid colony formation is mainly mediated through TNFR-p55, although TNFR-p75-mediated inhibition could be observed on progenitors responsive to erythropoietin alone. Moreover, at low TNF-alpha concentrations (2 ng/mL), TNF-alpha stimulates interleukin-3-dependent in vitro growth of committed granulocyte-macrophage progenitor cells, whereas it potently inhibits erythroid progenitor cell proliferation, showing that one concentration of TNF-alpha can simultaneously and bidirectionally modulate interleukin-3-dependent growth of committed granulocyte-macrophage (stimulation) and erythroid progenitor cells (inhibition).

Partial purification and characterization of a factor for the enhancement of colony formation in vitro by myeloid progenitor cells.

We have purified a factor, hematopoietic promoting factor (HPF), from porcine kidney extract (PKE), which exhibits a promoting activity on granulocyte/macrophage (GM) colony and burst-forming-unit-erythroid (BFU-E)-derived colony formation by progenitors from murine bone marrow cells in vitro. The addition of HPF resulted in an enhancement of the GM colonies as well as BFU-E-derived colonies, but did not enhance the colony-forming-unit-erythroid (CFU-E)-derived colony formation. HPF was added to the BFU-E cultures together with cytokines, such as recombinant murine interleukin-3 (IL-3), recombinant murine GM colony-stimulating-factor (GM-CSF) and recombinant human G-CSF, which have all been shown to enhance BFU-E growth. The combination of HPF plus these cytokines resulted in an enhancement of benzidine negative colony formation in comparison to the case of each cytokine alone; however, no increase was found on BFU-E colony formation. HPF is able to enhance the granulopoiesis and erythropoiesis in vitro. And the synergistic activity of HPF is significantly affected by the presence of cytokines in the cultures.

Macrophage inflammatory protein (MIP)-1 beta abrogates the capacity of MIP-1 alpha to suppress myeloid progenitor cell growth.

The effects of recombinant murine macrophage inflammatory protein (MIP)-1 beta and MIP-2 on the suppressive activity of MIP-1 alpha were tested using colony formation by human and murine bone marrow burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte erythroid macrophage, megakaryocyte (CFU-GEMM), and colony-forming unit-granulocyte macrophage (CFU-GM) progenitor cells. MIP-1 beta, but not MIP-2, when added with MIP-1 alpha to cells, blocked the suppressive effects of MIP-1 alpha on both human and murine BFU-E, CFU-GEMM, and CFU-GM colony formation. Similar results were observed regardless of the early acting cytokines used: human rGM-CSF plus human rIL-3, and two recently described potent cytokines, a genetically engineered human rGM-CSF/IL-3 fusion protein and MGF, a c-kit ligand. The more potent the stimuli, the greater the suppressive activity noted. Pulse treatment of hu bone marrow cells with MIP-1 alpha at 4 degrees C for 1 h was as effective in inhibiting colony formation as continuous exposure of cells to MIP-1 alpha, and the pulsing effect with MIP-1 alpha could not be overcome by subsequent exposure of cells to MIP-1 beta. Also, pulse exposure of cells to MIP-1 beta blocked the activity of subsequently added MIP-1 alpha. For specificity, the action of a nonrelated myelosuppressive factor H-ferritin, was compared. MIP-1 alpha and H-ferritin were shown to act on similar target populations of early BFU-E, CFU-GEMM, and CFU-GM. MIP-1 beta did not block the suppressive activity of H-ferritin. Also, hemin and an inactive recombinant human H-ferritin mutein counteracted the suppressive effects of the wildtype H-ferritin molecule, but did not block the suppressive effects of MIP-1 alpha. These results show that MIP-1 beta's ability to block the action of MIP-1 alpha is specific. In addition, the results suggest that MIP-1 alpha and MIP-beta can, through rapid action, modulate early myeloid progenitor cell proliferation.