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• W2233125730 abstract "Premature intramedullary apoptosis of erythroid progenitors is an accepted paradigm for the ineffective erythropoiesis of β-thalassaemia major.( Yuan et al, 1993; Centis et al, 2000; Mathias et al, 2000) However, delayed maturation, associated with expression of cell cycle promoting and survival factors has been described in β-thalassaemia mouse models.(Libani et al, 2008) Here we report data that supports dysregulation of the cell cycle as a significant contributor to the ineffective erythropoiesis of β-thalassaemia major. CD34+ haemopoietic stem cells were obtained from the peripheral blood of five transfusion-dependent β-thalassemia major patients and seven healthy controls as described previously.(Forster et al, 2015) These enriched CD34+ cells were cultured in MethoCult H4435 Enriched media (StemCell Technologies, Vancouver, BC, Canada) containing Iscove's Modified Dulbecco's Medium, supplemented with fetal bovine serum, stem cell factor, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interleukin (IL) 3, IL 6 and erythropoietin at 37°C in air with 5% CO2 for 14 days. Excluding myeloid cells (<3·5% for both control and patient groups) cytospin morphology showed the mean percentage of proerythroblasts, basophilic, polychromatic and orthochromatic erythroblasts was 1%, 3%, 14% and 82% for the control group and 6%, 10%, 27% and 57% for the patient group (Fig 1A). These values were all significantly different (P = 0·0002, 0·0015, 0·0011 and <0·0001). The relative reduction in the mature erythroblast subpopulation in the patient group is suggestive of a delay in maturation as opposed to increased cell death. This pattern has been observed previously both in vitro (Mathias et al, 2000; Forster et al, 2015) and in vivo in the assessment of bone marrow aspirates (Yuan et al, 1993; Centis et al, 2000). Apoptosis was assessed by three flow cytometric methods: (i) Annexin V binding and propidium iodide exclusion using the fluorescein isothicyanate (FITC) Annexin V Apoptosis Detection Kit II (BD Biosciences, San Jose, CA, USA), (ii) active caspase 3 was measured with the phycoerythrin Active Caspase 3 Apoptosis Kit (BD Biosciences) and (iii) internucleosomal cleavage of DNA was determined using the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay with the APO-BRDU Kit (BD Biosciences). We elected to use a range of assays to assess apoptosis, as controversies over the different methodologies have been described in the literature. Firstly, β-thalassaemic cells present with increased phosphatidyl serine (PS) exposure, (Libani et al, 2008) secondly, caspase-3, a key protease in the apoptotic caspase cascade, also has a non-apoptotic role in erythroid maturation during erythropoiesis (Carlile et al, 2004) and finally, large numbers of foetal erythroblasts are TUNEL positive but PS negative.(Hristoskova et al, 2003). Although there were differences between methodologies, when cells from thalassaemic patients were compared with cells from healthy controls there was no statistically significant difference in the level of apoptosis (Fig 1B). This supports the results from our previous in vitro gene expression study (Forster et al, 2015) where during the later stages of erythroblast maturation, while genes with apoptotic properties were expressed, a clear apoptotic signal was not identified in the thalassaemic cells. These results differ from previous work by Mathias et al (2000), who showed increased apoptosis in both marrow aspirates and erythroid cell cultures. However, their erythroid culture analysis only included three samples and, with the significant heterogeneity observed in this study in both control and patient groups, these variations may not have been detected due to low sample size. Differing culture methods may also have contributed to the non-concurring observations. Cell cycle stages were measured by flow cytometry using propidium iodide (PI)/RNase Staining Buffer and Alexa Fluor 647 Rat anti-Histone H3 (pS28) (BD Biosciences). The cell cycle, as assessed by PI incorporation, was divided into G0/G1, S and G2/M phases. G2 and M could then be further distinguished by the presence of the phosphorylated Histone H3 in M-phase cells (Fig 1C). The percentage of cells in the G0/G1 and S-phases was not significantly elevated in patients while the percentage of cells in M-phase was reduced (P = 0·01) (Fig 1D). Median percentages were 63·6% for control and 71·3% for thalassaemic cells in G0/G1 phase, 10·6% for control and 12·8% for thalassaemic cells in S phase, 20·9% for control and 17·6% for thalassaemic cells in G2 phase and 6·3% for control and 1·7% for thalassaemic cells in M phase. The proportion of cells in different phases of the cell cycle for the controls agrees with other in vitro cell cycle work on erythroblasts.(Fibach & Rachmilewitz, 1993; Wojda et al, 2002) Increased S-phase proportions have been reported in β-thalassaemia (Libani et al, 2008) but not a change in M-phase. This may be a result of the methodology used in these studies, which did not separate the G2 and M phases. The decreased proportion of cells in M-phase may indicate a failure to enter mitosis at later stages of erythroid development. Proliferation was determined by flow cytometry using FITC Mouse Anti-Human Ki-67 (BD Biosciences) and measured by the proportion of cells expressing Ki-67. This protein is expressed when the cell is in cycle and is rapidly downregulated when the cell leaves the cell cycle. A further subcategory of strongly expressing cells was defined as Ki-67High (Fig 1E). By both measures the patient group had more proliferating cells than the control group (Fig 1F). Median Ki-67 + was 49·9% for controls and 69·9% for patients (P = 0·012) while median Ki-67High was 8·9 for controls and 13·2 for patients (P = 0·046). Proliferation is increased in vivo in β-thalassaemia due to the increased erythropoietin drive, a function of the anaemia and ineffective erythropoiesis. However, here we observe an increase in Ki-67 expression in standardized culture conditions, which implies it is an intrinsic property of the thalassaemic cells. Proliferation is higher in more immature erythroblasts (Wojda et al, 2002) with a higher proportion in cycle earlier in maturity.(Dai et al, 2000). In conclusion, this in vitro study showed no demonstrable increase in apoptosis. In contrast this study showed increased Ki-67 expression consistent with an increased proportion of cells in the cell cycle, together with a reduction in the proportion of cells in M phase. This combination of features would be consistent with dysregulation of the cell cycle as a contributing factor to the ineffective erythropoiesis characteristic of β- thalassaemia major. The authors would like to acknowledge PathWest Laboratory Medicine WA for providing laboratory space and financial support of this project. We would also like to acknowledge the School of Pathology and Laboratory Medicine and The University of Western Australia for providing Luke Forster's PhD scholarship. We are thankful for the statistical advice that the Department of Research at Sir Charles Gairdner Hospital provided for this project. Luke Forster: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing. Scott Cornwall: Collection and/or assembly of data, data analysis and interpretation. Jill Finlayson: Conception and design, data analysis and interpretation critical revision of manuscript. Reza Ghassemifar: Conception and design, data interpretation and critical revision of manuscript." @default.
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• W2233125730 date "2016-01-13" @default.
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• W2233125730 title "Cell cycle, proliferation and apoptosis in erythroblasts cultured from patients with β-thalassaemia major" @default.
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