Number Of Burst-forming Units-erythroid Research Articles

A Pharmacologic Approach Using Pomalidomide and Dexamethasone to Study Stage-Specific Regulation of Erythroid Progenitors

The erythroid progenitors, burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E), control red cell production through the integration of a variety of self-renewal and differentiation signals. The identification of these signals and their effects on erythroid progenitor populations are of therapeutic interest for human disorders characterized by decreased red cell production such as Diamond Blackfan Anemia (DBA). We have previously documented that pomalidomide acts at the BFU-E stage and dexamethasone at the CFU-E stage in in vitro models of human erythropoiesis. Therefore, these two agents represent potential pharmacologic interventions for the treatment of human diseases with disordered erythropoiesis. Here, we studied the mechanisms by which pomalidomide and dexamethasone enhance erythroid progenitor numbers in cord blood and adult bone marrow-derived CD34+ hematopoietic stem and progenitor cells (HSPC).Pomalidomide increased adult and fetal BFU-E numbers in both serum-free and serum-containing in vitro erythroid culture systems. CFU-E numbers and terminal erythroblasts were also transiently attenuated suggesting that pomalidomide mediates increased BFU-E in part through delayed progression to erythropoietin-sensitive progenitors and precursor stages. Transforming growth factor-β (TGF-β) has previously been shown to regulate the transition from BFU-E to CFU-E, and therefore, we investigated whether pomalidomide modulates TGF-β signaling. We noted accelerated differentiation as assessed by glycophorin A (GPA) expression of CD34+ HSPCs treated with TGF-β1, and the addition of pomalidomide completely abrogated TGF-β1 mediated differentiation. This effect was not due to inhibition of TGF-β1 receptors or kinases as treatment of NIH 3T3 cells with TGF-β1 ± pomalidomide failed to prevent SMAD2 phosphorylation. Only CD34+ HSPCs differentiated in the presence of pomalidomide exhibited reduced sensitivity to TGF-β1. These results suggest that pomalidomide alters erythroid and/or earlier hematopoietic progenitor differentiation to enrich a population of TGF-β1 insensitive cells and/or affects the activity of downstream TGF-β1 effectors.Dexamethasone, in contrast to pomalidomide, was found to specifically increase the number of CFU-E progenitors. Elevated CFU-E self-renewal was associated with an accumulation of cells in G0/G1 of the cell cycle, which suggests that alterations to cell cycle progression may contribute to the regulation of self-renewal genes in CFU-E. In contrast to adult-derived CFU-E, dexamethasone failed to affect CFU-E numbers or cell cycle status in cord blood derived CFU-Es. We investigated the expression of glucocorticoid receptor isoforms (GRα and GRβ) in erythroid progenitors, which revealed that BFU-E and CFU-E differentially express GR isoforms. BFU-E expressed comparable levels of GRα and the hypothesized dominant-negative GRβ, where as only GRα transcript was measured in CFU-E. These expression patterns were similar between adult and cord blood-derived progenitors. Despite similar GR levels, adult-derived erythroid progenitors, but not cord blood derived progenitors, were shown to translocate GRα to the nucleus after a 24hr pulse of dexamethasone suggesting that post-translational mechanisms may govern dexamethasone response in erythroid progenitors.These findings strongly indicate that pharmacologic modulation of the TGF-β and glucocorticoid-signaling pathways can effectively stimulate erythroid progenitor self-renewal. We are currently elucidating specific targets for pomalidomide and dexamethasone in BFU-E and CFU-E, respectively, through proteomics and transcriptome approaches, and investigating the efficacy of mono- and combination therapies using peripheral blood-derived CD34+ HSPCs from DBA patients to study their therapeutic efficacy. DisclosuresNo relevant conflicts of interest to declare.

Single Cell Resolution of Glucocorticoid Effects on Erythroid Progenitor Cells

To generate the billions of new erythrocytes required on a daily basis, erythroid progenitor cells must exponentially increase in number before undergoing terminal differentiation. A limited number of cell divisions occur during erythropoietin (EPO)-regulated erythroid terminal differentiation, but the principal regulation of erythroid transit-amplification occurs earlier in erythropoiesis between the burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E) stages of development. The importance of this EPO-independent early erythropoietic process is highlighted in Diamond-Blackfan Anemia (DBA). DBA is characterized by pure red cell aplasia, loss of BFU-E and CFU-E progenitors in the bone marrow, and severe anemia despite high circulating EPO levels. The only known effective medical therapy for DBA also provides insight into the regulation of erythroid transit-amplification. In patients responsive to glucocorticoid treatment, there are increased numbers of BFU-E and CFU-E progenitors in the bone marrow, and ex vivo culture studies indicate that the synthetic glucocorticoid dexamethasone (Dex) predominantly increases proliferative capacity of BFU-Es, with minimal effect on proliferative capacity of CFU-Es. These findings led to a prevailing model that glucocorticoids increase BFU-E proliferative capacity by stimulating several rounds of self-renewal cell divisions. However, a limitation of this model is that there is no mechanistic explanation for how BFU-Es regulate the fate choice of undergoing a self-renewal cell division versus a differentiation cell division in the presence or absence of glucocorticoids. In this study, we address this question by examining progression of early erythroid progenitor development at single cell resolution, and subsequently elucidate the true mechanistic nature of glucocorticoid-induced erythroid progenitor proliferative capacity amplification. By performing single cell transcriptome profiling (scRNAseq) of primary-isolated mouse fetal liver BFU-Es, CFU-Es, and their developmental intermediates, we identify a continuum of transcriptomic states during erythroid transit-amplification when performing principle component analysis (PCA) on transcriptomes of individual cells. We show that ex vivo culture of primary-isolated BFU-Es in serum free media supplemented with stem cell factor, insulin-like growth factor 1, and EPO results in developmental progression along the transcriptome continuum when performing scRNAseq and PCA on cultured BFU-Es. The addition of Dex into this culture system does not result in self-renewal of BFU-Es at the transcriptome level, but rather still results in developmental progression, albeit to less of a degree per cell division than BFU-Es cultured without Dex. We additionally show that the continuum of transcriptome states in erythroid transit-amplification is reflective of a continuum of functional states, with developmental progression characterized by decreasing proliferative capacity and decreasing glucocorticoid-responsiveness. Lastly, through manual separation of daughter cells resulting from a BFU-E cell division, we demonstrate that BFU-E cell division is a symmetric process at the transcriptome level, both with and without the addition of Dex. Our results clarify the nature of how glucocorticoids amplify BFU-E proliferative capacity. As opposed to stimulating a finite number of BFU-E self-renewal cell divisions, glucocorticoids decrease the extent of progression through the erythroid transit-amplifying developmental continuum per cell division. Thus, a decreased rate of progression through the developmental continuum is associated with an increased number of transit-amplifying cell divisions prior to terminal differentiation. These findings are important not only for the rational development of glucocorticoid-alternatives for treating DBA, but also for all bone marrow failure syndromes characterized by progenitor cell hypoplasia. DisclosuresNo relevant conflicts of interest to declare.

The orally bioavailable MDM2 antagonist RG7112 and pegylated interferon α 2a target JAK2V617F-positive progenitor and stem cells

The Philadelphia chromosomal-negative chronic myeloproliferative neoplasms (MPNs) originate at the level of the hematopoietic stem cell (HSC). The protracted clinical course of the MPNs has limited the use of potentially toxic treatment modalities, which may eliminate the responsible malignant clone. Treatment with low doses of RG7112, an orally available small-molecule inhibitor of p53-MDM2, both alone and combined with pegylated interferon α 2a (Peg-IFNα 2a), significantly decreased MPN colony-forming unit-granulocyte macrophage and burst-forming unit-erythroid numbers and preferentially eliminated the total number of JAKV617F(+) MPN hematopoietic progenitor cells. The effects of RG7112 and Peg-IFNα 2a on MPN progenitor cells were dependent on blocking p53-MDM2 interactions and activating the p53 pathway, thereby increasing MPN CD34(+) cell apoptosis. Treatment of polycythemia vera (PV) and primary myelofibrosis (PMF) CD34(+) cells with low doses of RG7112 and Peg-IFNα 2a before their transplantation into immune-deficient mice decreased the degree of donor-derived chimerism as well as the JAK2V617F allele burden, indicating that these drugs can each alone or in combination deplete MPN HSCs. These results provide a rationale for the use of combinations of low doses of RG7112 and Peg-IFNα 2a for the treatment of PV or PMF patients with the intent of altering their natural history.

Abstract 426: In-field head and neck irradiation radiosensitivity of FancD2-/- mice and bystander effect reduction of distant bone marrow hematopoietic progenitor cells.

Abstract Background: Irradiation induced normal tissue toxicity limits effective radiotherapy of individuals with DNA repair defects including Fanconi anemia. We quantitated in-field head and neck tissue response in irradiated Fanconi anemia knockout mice, FancD2-/-, compared to wild type, FancD2+/+ & heterozygous FancD2+/- mice. We also assessed bystander effect of head and neck irradiation on distant bone marrow hematopoietic progenitor cell numbers. Method: Adult 10-12wk old FevB background FancD2+/+, +/- and -/- mice were irradiated to the head & neck to 24Gy, 26Gy, 28Gy or 30Gy (n=3) shielding all tissue below the cervical spine. On day 2 or 5 post irradiation, mice were sacrificed and tongue tissue and one femur excised for histopathology. Bone marrow of the contralateral femur was plated for colony assay. Colony forming unit-granulocyte macrophage (CFU-GM), burst forming unit erythroid (BFU-E), and colony forming unit-granulocyte-erythroid-megakaryocyte-monocyte (CFU-GEMM) numbers were counted & each genotype compared to its unirradiated control. H&E stained tongue sections were scored for oral mucositis. Results: Both FancD2-/- & FancD2+/- mice were radiosensitive to lower doses compared to FancD2+/+ mice measured as percent ulceration of the squamous layer of tongue epithelium 5 days post irradiation. Both FancD2+/- and FancD2-/- mice had significantly higher ulceration after 26Gy (52.8±6.5%, p=0.0014 and and 59.4±3.4%, p=0.0001, respectively) compared to wild type FancD2+/+ (21.7±4.7%). At the lower dose of 24Gy, FancD2+/- and -/- had higher percent ulceration (p=0.0001 and p=0.0003) than FancD2+/+ mice irradiated to even higher doses of 26Gy and 28Gy. FancD2+/+ mice had high levels of ulceration after 28Gy (p=0.2355). While there was no difference in distant bone marrow cellularity between irradiated groups, both FancD2-/- & FancD2+/- showed bystander reduction in colony forming cells. CFU-GM were significantly reduced at 2 days after 26Gy in FancD2+/- mice compared to unirradiated controls (p=0.007). FancD2 -/- mice had decreased BFU-E after 24Gy (p=0.009) & CFU-GEMM after 26Gy (p=0.048) and reduced BFU-E numbers after 26Gy (p=0.031). FancD2-/- mice had significantly less CFU-GM after either 26Gy or 28Gy (p=0.017 and p=0.028, respectively). FancD2-/- & +/- mice, at 5 days after 26Gy, had less CFU-GM (p=0.001 and 0.012, respectively) and CFU-GEMM (p=0.014 and p=0.004, respectively) compared to unirradiated controls. There was no reduction in CFU-GM, BFU-E or CFU-GEMM in wild type FancD2+/+mice after 28 or 30Gy. Conclusion: Biallelic or monoallelic loss of the FA gene renders mice radiosensitive to in-field head and neck irradiation compared to wild type mice, and is accompanied by a distant femur marrow bystander effect including reduction of colony forming unit. Supported by NIAID U19-A 1068021 and FA Research Foundation Citation Format: Hebist Berhane, Julie Goff, Ronny Kalash, Michael W. Epperly, Tracy Dixon, Donna Shields, Darcy Franicola, Joel S. Greenberger. In-field head and neck irradiation radiosensitivity of FancD2-/- mice and bystander effect reduction of distant bone marrow hematopoietic progenitor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 426. doi:10.1158/1538-7445.AM2013-426

Protective effect of Dammarane Sapogenins against chemotherapy-induced myelosuppression in mice

Chemotherapy is the most common way to treat malignancies, but myelosuppression, one of its common side-effects, is a formidable problem. The present study described the protective role of dammarane sapogenins (DS), an active fraction from oriental ginseng, on myelosuppression induced by cyclophosphamide (CP) in mice. DS was orally administered at different dosages (37.5, 75, and 150 mg/kg) for 10 d after CP administration (200 mg/kg intraperitoneally). The results showed that DS increased the number of white blood cells (WBC) on day 3 and day 7 (P < 0.05), such that WBC levels were increased by 105.7 ± 29.5% at 75 mg/kg of DS on day 3 (P < 0.05, compared with the CP group). Similar results were observed in red blood cells and platelets in DS-treated groups. The colony-forming assay demonstrated that the depressed numbers of CFU-GM (colony-forming unit-granulocyte and macrophage), CFU-E (colony-forming unit-erythroid), BFU-E (burst-forming unit-erythroid), CFU-Meg (colony-forming unit-megakaryocyte) and CFU-GEMM (colony-forming unit-granulocyte, -erythrocyte, -monocyte and -megakaryocyte) induced by CP were significantly reversed after DS treatment. Moreover, the ameliorative effect of DS on myelosuppression was also observed in the femur by hematoxylin/eosin staining. In DS-treated groups, ConA-induced splenocyte proliferation was enhanced significantly at all the doses (37.5, 75, 150 mg/kg) on day 3 at the rate of 50.3 ± 8.0%, 77.6 ± 8.5% and 44.5 ± 8.4%, respectively, while lipopolysaccharide-induced proliferation was increased mainly on day 7 (P < 0.01), with an increased rate of 39.8 ± 5.6%, 34.9 ± 6.6% and 38.3 ± 7.3%, respectively. The thymus index was also markedly increased by 70.4% and 36.6% at 75 mg/kg on days 3 and 7, respectively, as compared with the CP group. In summary, DS has a protective function against CP-induced myelosuppression. Its mechanism might be related to stimulating hematopoiesis recovery, as well as enhancing the immunological function.

BCR-ABL but Not JAK2 V617F Inhibits Erythropoiesis through the Ras Signal by Inducing p21CIP1/WAF1

BCR-ABL is a causative tyrosine kinase (TK) of chronic myelogenous leukemia (CML). In CML patients, although myeloid cells are remarkably proliferating, erythroid cells are rather decreased and anemia is commonly observed. This phenotype is quite different from that observed in polycythemia vera (PV) caused by JAK2 V617F, whereas both oncogenic TKs activate common downstream molecules at the level of hematopoietic stem cells (HSCs). To clarify this mechanism, we investigated the effects of BCR-ABL and JAK2 V617F on erythropoiesis. Enforced expression of BCR-ABL but not of JAK2 V617F in murine LSK (Lineage(-)Sca-1(hi)CD117(hi)) cells inhibited the development of erythroid cells. Among several signaling molecules downstream of BCR-ABL, an active mutant of N-Ras (N-RasE12) but not of STAT5 or phosphatidylinositol 3-kinase (PI3-K) inhibited erythropoiesis, while N-RasE12 enhanced the development of myeloid cells. BCR-ABL activated Ras signal more intensely than JAK2 V617F, and inhibition of Ras by manumycin A, a farnesyltransferase inhibitor, ameliorated erythroid colony formation of CML cells. As for the mechanisms of Ras-induced suppression of erythropoiesis, we found that GATA-1, an erythroid-specific transcription factor, blocked Ras-mediated mitogenic signaling at the level of MEK through the direct interaction. Furthermore, enforced expression of N-RasE12 in LSK cells derived from p53-, p16(INK4a)/p19(ARF)-, and p21(CIP1/WAF1)-null/wild-type mice revealed that suppressed erythroid cell growth by N-RasE12 was restored only by p21(CIP1/WAF1) deficiency, indicating that a cyclin-dependent kinase (CDK) inhibitor, p21(CIP1/WAF1), plays crucial roles in Ras-induced suppression of erythropoiesis. These data would, at least partly, explain why respective oncogenic TKs cause different disease phenotypes.

Pure red-cell aplasia as the presenting feature of the carcionoid tumor of the thymus: case report

Acquired pure red-cell aplasia (PRCA) is an uncommon disorder of erythrocytopoiesis that can develop in association with thymic tumors. We present the very rare case of a severely anemic 62-year-old man with PRCA and a concurrent neuroendocrine carcinoid tumor of the thymus. The anterior mediastinal thymus tumor was completely excised, and following histological and immunohistochemical analyses (showing positive staining for cytokeratin, chromogranin A, synaptophysin, and neuron-specific enolase) the diagnosis of a (grade I; T(1)N(0)M(0)) typical carcinoid tumor of the thymus was made. Postoperatively the anemia persisted despite no signs of residual tumor on CT chest. A hematological work up found: normocellularity with <0.5% erythroblasts and preserved megakaryocytopoiesis and granulocytopoiesis in a trephine biopsy; reduced numbers of Colony Forming Unit Erythroid (CFU-E) and normal numbers of Burst-Forming Unit Erythroid (BFU-E) in bone marrow colony-forming assays; a markedly increased level of serum erythropoietin; normal T and B-cell numbers with a normal CD4/CD8 ratio; and no clonal T-cell receptor -gamma and -delta gene rearrangement) The patient responded favorably to a therapeutic trial of glucocorticoid immunosuppressive treatment (prednisone 1 mg/kg/day) with a normalization of the reticulocyte count and hematocrit, suggesting an immunologic mechanism for the PRCA. Though the exact mechanisms underlying the association between the PRCA and the carcinoid tumor of the thymus remain unknown.

Hypoxia-inducible Factor-1 Deficiency Results in Dysregulated Erythropoiesis Signaling and Iron Homeostasis in Mouse Development

Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of genes whose products play critical roles in energy metabolism, erythropoiesis, angiogenesis, and cell survival. Limited information is available concerning its function in mammalian hematopoiesis. Previous studies have demonstrated that homozygosity for a targeted null mutation in the Hif1alpha gene, which encodes the hypoxia-responsive alpha subunit of HIF-1, causes cardiac, vascular, and neural malformations resulting in lethality by embryonic day 10.5 (E10.5). This study revealed reduced myeloid multilineage and committed erythroid progenitors in HIF-1alpha-deficient embryos, as well as decreased hemoglobin content in erythroid colonies from HIF-1alpha-deficient yolk sacs at E9.5. Dysregulation of erythropoietin (Epo) signaling was evident from a significant decrease in mRNA levels of Epo receptor (EpoR) in Hif1alpha-/- yolk sac as well as Epo and EpoR mRNA in Hif1alpha-/- embryos. The erythropoietic defects in HIF-1alpha-deficient erythroid colonies could not be corrected by cytokines, such as vascular endothelial growth factor and Epo, but were ameliorated by Fe-SIH, a compound delivering iron into cells independently of iron transport proteins. Consistent with profound defects in iron homeostasis, Hif1alpha-/- yolk sac and/or embryos demonstrated aberrant mRNA levels of hepcidin, Fpn1, Irp1, and frascati. We conclude that dysregulated expression of genes encoding Epo, EpoR, and iron regulatory proteins contributes to defective erythropoiesis in Hif1alpha-/- yolk sacs. These results identify a novel role for HIF-1 in the regulation of iron homeostasis and reveal unexpected regulatory differences in Epo/EpoR signaling in yolk sac and embryonic erythropoiesis.

Effects of an Angiotensin II Receptor Antagonist and Angiotensin-Converting Enzyme Inhibitors on Burst Forming Units-Erythroid in Chronic Hemodialysis Patients

Background: Angiotensin-converting enzyme (ACE) inhibitors have been reported to reduce the response to erythropoietin (EPO) administration in chronic hemodialysis patients, but the mechanism for this effect has not yet been clarified. To clarify the mechanism of ACE inhibitors- and angiotensin II type 1 (AT1) receptor antagonist-induced anemia in hemodialysis patients, we examined the effect of ACE inhibitors and AT1 receptor antagonist on burst-forming units-erythroid (BFU-E) in the peripheral blood of hemodialysis patients and healthy controls in vitro. Methods: Peripheral blood mononuclear cells (PBMNCs) were isolated by gradient centrifugation from 10 patients on regular hemodialysis and 7 healthy control volunteers. A colony assay of hematopoietic progenitors was performed using the methylcellulose culture system. PBMNCs of 1 or 2 × 10⁵ were plated in a medium containing EPO with various concentrations of ACE inhibitors or AT1 receptor antagonist and incubated for 14 days. Colonies of BFU-E were counted under an inverted microscope. Results: The PBMNCs from the chronic hemodialysis patients formed fewer BFU-Es than those from healthy volunteers. AT1 receptor antagonist in both healthy volunteers and hemodialysis patients suppressed the number of BFU-Es. The ACE inhibitors produced a smaller effect than the AT1 receptor antagonist. Conclusion: AT1 receptor blockade can directly inhibit erythropoiesis in vitro.

Erythroid Progenitor Cells from Pig Bone Marrow and Peripheral Blood

With the intention of using the pig as a large animal model in haematopoietic research, a clonal assay in methylcellulose was developed and the optimal conditions for raising erythroid progenitors from adult pig bone marrow (BM) and peripheral blood (PB) have been established. Progenitor cells were stimulated to proliferate and differentiate in vitro by growth factors containing leucocyte condition medium (LCM), and with recombinant human erythropoietin (rhEpo). The number of PB BFU-E (burst forming units – erythroid) directly depended on the concentration of LCM, but BM BFU-E were not dependent on LCM. Both CFU-E (colony forming units – erythroid) and BFU-E were rhEpo dependent. Despite relatively high but expected individual variations, the mean number of colonies, as well as the functional characteristics of progenitor cells investigated, were similar to those of miniature pigs and some other mammals.

EFFECTS OF CYCLOSPORINE IMMUNOSUPPRESSIVE THERAPY ON HUMAN HEMATOPOIESIS IN LIVER TRANSPLANTED PATIENTS

372 This study investigates the patterns of hematopoietic abnormalities in liver transplanted (OLTX) patients who were treated with cyclosporine A (CyA) and steroids. Eight Pts with at least one of the following criteria were selected: Hb ≤ 10 gr/dl. WBC ≤ 3000/μl, Plts ≤ 100000/μl. Pts with bone marrow (BM) alterations due to known variables were excluded. The pts were at least six months post-op and the indications for OLTX were ETOH cirrhosis, primary sclerosing cholangitis and primary billary cirrhosis. BM cellularity was assessed by histology and three groups of pts were identified: normocellular (n=2), hypercellular (n=3) and hypocellular (n=3). BM aspirate smears were evaluated according to the French-American-British criteria and variable grades of myeloid (7 pts) and erythroid (6 pts) dysplastic features were observed. BM aspirates from the enrolled pts and healthy controls were cultured in growth factor/cytokine-enriched methylcellulose media in the presence of increasing concentrations of CyA (0, 50, 300, 1000 ng/ml). In the plain cultures (without CyA), hypocellular BMs developed only a few Burst Forming Units-Erythroid (BFU-E), while in normocellular BMs the overall number of BFU-E remained similar to healthy controls. On the contrary, in the hypercellular group the number of BFU-E was significantly increased (210.6 ± 78.5 v.s. 96.6 ± 41.5, p < 0.05). The addition of CyA reduced significantly, when compared to plain cultures, the number of BFU-E in hypocellular group (21 ± 8.4 v.s. 37.7 ± 15.9, p< 0.05), hypercellular group (158 ± 59.3 v.s. 210.6 ± 78.5, p < 0.05) and in healthy controls. In contrast, the number of BFU-E in normocellular group was comparable to those in the plain cultures. We did not observe remarkable changes in Granulocyte-Monocyte Colony Forming Units in the presence of CyA. In conclusion, we observed a meaningful depressing effect of CyA on erythropoiesis of pts with histological abnormalities of the BM cellularity. The increased number of erythroblast-committed cells (CD34+/71+ 2.6% ± 0.7 v.s. 06% ± 0.3 in healthy controls), demonstrated by cytoflowmetry, did not correlate with peripheral blood results which were consistent with anemia and/or leucopenia. These data together with the constant finding of erythro-myelodysplasia may indicate an inefficient erythropoiesis in the BM of OLTX pts. The results of our study suggest that the determination of BM cellularity may predict the effect of CyA on erythropoiesis where no clear causes of anemia in post OLTX can be identified. The frequent presence of BM dysplasia and altered percentage of pluripotent stem cells and erythroblast committed cells do not seem to have clear clinical significance.

In vitro culture growth of erythroid progenitors and serum erythropoietin assay in the differential diagnosis of polycythaemia.

We assessed the in vitro culture growth of erythroid progenitors [burst forming unit-erythroid (BFU-E)] and serum erythropoietin (EPO) levels in different groups of polycythaemia to determine the discriminative power in differential diagnosis of polycythaemia. We used the methylcellulose culture technique to study the growth of endogenous erythroid colonies (EECs) and EPO-dependent BFU-E from bone marrow (BM) and/or peripheral blood (PB) cells from 40 patients with polycythaemia vera (PV), 13 with secondary polycythaemia (SP), 19 with pure erythrocytosis (PE), five with PE and PV evolution later (PE-PV), and 12 with relative polycythaemia (RP). The serum EPO levels were measured by radioimmunoassay before treatment in 47 patients, 23 SP patients, 19 PE patients, five PE-PV patients and 16 RP patients, as well as after treatment in 38 PV patients, five PE-PV patients and 12 PE patients. The results of the erythroid progenitor culture assay showed that the numbers of EPO-dependent BFU-E in BM did not differ significantly among groups. The PB BFU-E were significantly higher in PV than in SP or PE, and no statistical difference were found among patients with SP, PE and RP. There was a correlation between BM BFU-E and PB BFU-E in the individual PV and PE patients. EECs were present in all BM and PB cultures of untreated and phlebotomy-treated PV and PE-PV patients, but were absent in 6 of 17 PV patients who had received cytotoxic therapy. EECs were not found in SP, PE and RP. PB could substitute for BM in the EEC or the BFU-E assay. Both pretreatment and post-treatment serum EPO levels of PV and PE-PV were similar, which were significantly lower than SP, PE or RP. The serum EPO levels in treated PV or PE-PV patients who had normal haematocrit values were not significantly different from those in untreated patients. In contrast, the phlebotomy-treated PE patients had significantly higher serum EPO values than untreated PE patients. In the differentiation between PV and PE, the sensitivity, specificity, positive predictive value and negative predictive value of post-treatment serum EPO levels at a cut-off level of < or = 9 UL-1 were 74%, 92% and 52% respectively. The discriminative power of post-phlebotomy serum EPO levels was even higher with a positive predictive value of 80% and negative predictive value of 92% for the prediction of PV evolution in patients with pure erythrocytosis of unknown origin. The present study showed that apart from EEC assay, the post-phlebotomy serum EPO level was a sensitive and specific parameter in the differential diagnosis of polycythaemia, in particular for the identification of PV among patients with unclassifiable polycythaemia.

The Assessment of the Hematopoietic Reservoir After Immunosuppressive Therapy or Bone Marrow Transplantation in Severe Aplastic Anemia

AbstractWe investigated the hematopoietic reservoir in 43 severe aplastic anemia (SAA) patients following immunosuppression (IS) (n = 15) or bone marrow transplantation (BMT) (n = 28), at a median interval of 5 years (range, 2-20) from treatment. All patients had normal blood counts, good marrow cellularity, and normal numbers of colony forming unit-granulocyte macrophages (CFU-GM). Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte erythroid megakaryocyte macrophages (CFU-GEMM) numbers were reduced when compared with normal controls. However, the most pronounced defect was observed at the level of long-term culture-initiating cells (LTC-IC), which significantly differed from controls (P < .00001) both for IS and BMT patients. Their number did not improve with time and was not affected by transplant or treatment-related variables. When IS patients were compared with BMT we found comparable numbers of CFU-GEMM (P = .8) and LTC-IC (P = .9), but lower numbers of BFU-E and CFU-GM (P = .05 and P = .004, respectively), suggestive of a persistent suppressive mechanism. These data indicate that LTC-IC numbers are severely reduced in BMT and IS patients, contradicting the common belief that the former are fully reconstituted as compared with the latter. In addition, the number of mature cells and committed progenitors does not seem to reflect the real size of the hematopoietic reservoir and few stem cells may be sufficient to guarantee normal hematopoiesis long term.

The Assessment of the Hematopoietic Reservoir After Immunosuppressive Therapy or Bone Marrow Transplantation in Severe Aplastic Anemia

We investigated the hematopoietic reservoir in 43 severe aplastic anemia (SAA) patients following immunosuppression (IS) (n = 15) or bone marrow transplantation (BMT) (n = 28), at a median interval of 5 years (range, 2-20) from treatment. All patients had normal blood counts, good marrow cellularity, and normal numbers of colony forming unit-granulocyte macrophages (CFU-GM). Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte erythroid megakaryocyte macrophages (CFU-GEMM) numbers were reduced when compared with normal controls. However, the most pronounced defect was observed at the level of long-term culture-initiating cells (LTC-IC), which significantly differed from controls (P &lt; .00001) both for IS and BMT patients. Their number did not improve with time and was not affected by transplant or treatment-related variables. When IS patients were compared with BMT we found comparable numbers of CFU-GEMM (P = .8) and LTC-IC (P = .9), but lower numbers of BFU-E and CFU-GM (P = .05 and P = .004, respectively), suggestive of a persistent suppressive mechanism. These data indicate that LTC-IC numbers are severely reduced in BMT and IS patients, contradicting the common belief that the former are fully reconstituted as compared with the latter. In addition, the number of mature cells and committed progenitors does not seem to reflect the real size of the hematopoietic reservoir and few stem cells may be sufficient to guarantee normal hematopoiesis long term.

The Supportive Effects of Erythropoietin and Mast Cell Growth Factor on CD34+/CD36- Sorted Bone Marrow Cells of Myelodysplasia Patients

In the present study, we analyzed the capacity of CD34+/CD36- sorted bone marrow cells of myelodysplasia patients (n = 4) to differentiate along the erythroid lineage in the presence of erythropoietin (Epo) and mast cell growth factor (MGF). Two subgroups could be identified. In 6 patients, a normal number of burst-forming units-erythroid (BFU-Es) were cultured from CD34+/CD36- sorted cells. Cells from these patients did have the capacity to differentiate to colony-forming units-erythroid (CFU-Es) progenitors in cell suspension cultures with Epo plus MGF followed by Epo in the culture assay. Moreover, the cells became CD34-/CD36+/gly-cophorin A (GpA)+ after 7 days of culture with Epo plus MGF, a pattern comparable to that of normal progenitors. In contrast, in 8 patients, a different pattern was observed. No BFU-Es or a low number of BFU-Es were cultured from the CD34+/CD36- sorted cell fraction that was, in most of the cases, incapable of differentiating to CFU-E progenitors. Flow cytometry of the sorted population showed that, after 7 days of culture with Epo plus MGF, a high proportion of CD34+/CD36- cells persisted, whereas a low proportion of cells became CD34-/CD36+/GpA+. The unresponsiveness is not caused by the used growth factor combination, because the addition of interleukin-3 did not correct the defect. Evi-1 expression was studied in 9 cases to show whether an aberrant Evi-1 expression correlates with a disturbed erythroid development. Evi-1 expression was shown in 4 of 9 cases, whereas 3 of 9 cases did have a disturbed erythroid differentiation. In summary, the results show that the defects in the erythroid development in a subpopulation of patients with myelodysplasia is localized at an early stage of the erythroid differentiation and is associated with the persistent expression of the CD34 antigen and, in some cases, with the expression of Evi-1.

Clonogenic haematopoietic progenitor assays from clinically normal horses

Clonal assays for haematopoietic progenitors were performed on mononuclear cells isolated from bone marrow samples collected from the sternebrae of normal horses. Colony-forming units-erythroid (CFUE), burst-forming units-erythroid (BFU-E), colony-forming units-granulocyte/monocyte (CFU-GM) and colony-forming units-fibroblastic (CFU-F) were assayed, and reference values for clinically normal horses were established. The mean numbers of CFU-E, BFU-E and CFU-GM per 5 × 104 cells were 329 ± 48, 30 ± 5 and 131 ± 13, respective. The mean number of CFU-F was 49 ± 6 per 2 ± 10 cells. The numbers of CFU-E and BFU-E were linearly related to the concentrations of fetal bovine serum (FBS), bovine serum albumin (BSA) and the cell number plated. The number of CFU-E were increased in the presence of erythropoietin (EPO) but its presence was not required for colony growth. BFU-E had an absolute requirement for EPO but the number of BFU-E was not linearly related to dose of EPO. Methods for clonal assays of equine hematopoietic progenitors are described.

Accelerated Reconstitution of Platelets and Erythrocytes After Syngeneic Transplantation of Bone Marrow Cells Derived From Thrombopoietin Pretreated Donor Mice

The recent cloning of the ligand of the c-Mpl hematopoietin receptor has indicated a major role for this cytokine in the development of megakaryocytes. In this study we have applied c-Mpl ligand (thrombopoietin [TPO]) in the setting of syngeneic transplantation in an attempt to accelerate the reconstitution of platelets. Donor mice were treated with 20 kilounits (kU)/d TPO intraperitoneally (ip) for 5 days. This resulted in a 2.5-fold increment in platelet counts from 1,119 x 10(9)/L to 2,582 x 10(9)/L (mean, n = 7). Total numbers of hematopoietic progenitor cells in bone marrow (BM) and spleen, as assessed in a colony-forming unit-granulocyte erythroid monocyte macrophage (CFU-GEMM) colony assay (55.3 v 38.6 x 10(3) CFU/femur; 27.3 v 16.3 x 10(3) CFU/spleen, mean, n = 7) as well as total numbers of burst-forming unit-erythroid (BFU-E) (24.0 v 16.4 x 10(3)/femur; 10.2 v 1.9 x 10(3)/spleen, mean, n = 7), were significantly higher in TPO-treated donors than in saline-treated controls. Female Balb-C mice were lethally (8.5 Gy) irradiated and transplanted with 10(5) BM cells. After transplantation, groups of mice were treated with recombinant murine TPO at a dose of 20 to 30 kU/d ip or subcutaneously (SC) for 5 to 14 days. Using this dose and schedule, TPO did not stimulate the recovery of platelets in comparison with control animals transplanted with equal cell numbers but given vehicle alone. In other experiments, 10(5) BM cells were procured from TPO-treated donor mice and transplanted into lethally irradiated recipient mice. In comparison with animals transplanted with an equal number of BM cells derived from saline-treated controls, recipients of TPO-treated BM cells had significantly faster platelet recovery and higher platelet nadir counts (88 v 30 x 10(9)/L, mean, n = 20). Transplantation of TPO-treated BM cells also resulted in an accelerated recovery of erythrocytes and increased erythrocyte nadir counts (7.2 v 5.0 x 10(12)/L, mean, n = 20). At the day of platelet nadir (day 12 after transplantation) these animals had higher numbers of BFU-Es (770 v 422, mean, n = 5) in the marrow and also had higher reticulocyte counts (44 / 1000 v 8 / 1000 mean, n = 5) in the blood. Therefore, the accelerated recovery of erythrocytes may be a direct effect of TPO on erythropoiesis.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of total saponins of panax ginseng on hematopoietic progenitor cells in healthy humans and aplastic anemia patients

Ginseng is said to have beneficial effects on anemia. The proliferation effects of total saponins of Panax ginseng (TSPG) on hematopoietic progenitor cell in healthy individuals and 29 patients with aplastic anemia (AA) were observed through bone marrow cultures of burst forming unit-erythroid (BFU-E), colony forming unit-erythroid (CFU-E) and colony forming unit-granulocyte/macrophage (CFU-GM) in vitro compared with methyltestosterone (MT). The results suggest TSPG might prompt the proliferation of normal progenitor cells at a concentration of 20 µg/ml. The numbers of BFU-E,CFU-E and CFU-GM increased by 37.8±2.9%, 31.4±2.9% and 33.3±4.0% respectively overthe controls; furthermore TSPG was still useful to BFU-E, CFU-E growth without Epo in vitro, although the colony numbers were much lower. Otherwise MT was useless to CFU-GM. Of the 29 patients with AA, 14 who responded to MT showed sensitivity to TSPG in marrow culture (the rising rate of colony formation exceeded 30%), but immune-mediated AA (patient’s peripheral blood mononucleated cell suppressed normal hematopoiesis) and stem cell decreased AA (few of colonies were formed) showed almost no expression for TSPG activity because of the immunological suppression system and the absence of progenitors.

Relationship of burst-forming-unit-erythroid progenitors and their DNA-synthesis stage to fetal hemoglobin levels in hydroxyurea-treated patients with sickle cell anemia.

DNA-synthesis stage and total number of circulating burst-forming-units-erythroid (BFU-E) have been inversely correlated with hemoglobin F levels in the peripheral blood, as well as in the cells from the BFU-E-derived colonies obtained from homozygous sickle cell anemia (SS) patients during steady state. Similar studies in SS patients treated with cytotoxic agents have not been reported. However, regeneration of the erythroid marrow that follows the cytoreduction phase of chemotherapy has been suggested as one of the mechanisms of stimulation of fetal hemoglobin synthesis. Therefore, a longitudinal study of hemopoiesis in hydroxyurea-treated SS patients was conducted. Thirty-two sets of hemopoietic studies, including total circulating BFU-E and S-phase BFU-E, were obtained from three patients treated with hydroxyurea. A dose-dependent decrease in total BFU-E colonies occurred in peripheral blood of all three patients (r = -0.58, -0.85, and -0.97, respectively, with each P < 0.05). There was a strong positive correlation between hydroxyurea dose and fetal hemoglobin levels in two of the three patients who responded clinically (r = 0.89618 and 0.88632, respectively, with each P < 0.01). When data from all patients were combined (n = 32), there was a strong, inverse, linear relationship between total number of BFU-E and percentage S-phase BFU-E with fetal hemoglobin levels (r = -0.6649 and -0.7404, respectively, with each P < 0.0001). A stronger, curvilinear, multiple relationship was detected between total BFU-E and percentage S-phase BFU-E with fetal hemoglobin levels (R = 0.8351 and 0.8602 with each P < 0.0001).

Age-related alterations in erythroid and granulopoietic progenitors in Diamond-Blackfan anaemia.

Mechanisms involved in the erythroid failure characterizing Diamond-Blackfan anaemia (DBA) remain unidentified. The general consensus is that the defect is intrinsic to the marrow erythroid progenitor, but the target progenitor cell has not been precisely identified, and in vitro studies have revealed considerable heterogeneity between patients. In order to understand better the meaning of such a biological heterogeneity, we examined the in vitro response of erythroid progenitors CFU-E (colony-forming unit-erythroid) and BFU-E (burst-forming unit-erythroid) to erythropoietin (Epo), interleukin-3 (IL-3) and stem cell factor (SCF) in a large series of 24 patients from 1 month to over 20 years of age. Results of colony assays revealed a striking correlation between the age of the patient and the extent of the abnormalities detected in vitro. Therefore, despite profound anaemia, 80% (7/10) of the patients studied within 1 year of diagnosis had normal numbers of both CFU-E and BFU-E which exhibited a normal response to cytokines. In contrast, 12/14 patients followed up for more than 3 years had decreased numbers of erythroid progenitors, in seven cases associated with decreased colony-forming unit granulocyte-macrophage (CFU-GM). The number of CFU-E and BFU-E was not normalized even by the addition of high concentrations of combined Epo, IL-3 and SCF.(ABSTRACT TRUNCATED AT 250 WORDS)