Additional cell membrane alterations that occur in vivo, microcytosis was not evident [23,24]. Among the enucleated cells, hypochromia was obvious as several cells demonstrated an erythroid ghost-like appearance. However, the low number of enucleated beta-KD cells prevented more formal analyses of those cells. Enucleated cells were reportedly absent from ex vivo cultures of erythroblasts obtained from blood of thalassemia patients [25]. While we were unable to obtain the appropriate clinical samples for direct comparisons to be made between ex vivo cultures of betaKD versus thalassemia-major erythroblasts as part of this study, such comparisons should be made in laboratories where those cells are available. As demonstrated from other areas of biomedical research [26], there exists an urgent need for disease models using primary human cells or tissues. Considerable resources have been utilized in recent decades to develop and explore 11967625 murine models of hemoglobinopathies. Despite their robustness for understanding murine erythropoiesis in vivo, those disease models have not yet led to successful clinical trials for improving the health for patients with thalassemia. Differences between the kinetics of murine and human erythroblast differentiation [27], iron biology [7,28?0], and globin gene regulation [31] confound our ability to interpret the murine data in the context of clinical application. As a result, a considerable gap is developing between basic and clinical research for 1315463 beta-thalassemia. This study was undertaken to characterize the effects of betaglobin chain imbalances upon ex vivo erythroblast differentiation and survival using primary human cells. Our model for knockdown of beta-globin gene expression may also permit examinations of dysfunctional erythroblast heme metabolism or mitochondrial function caused by globin chain imbalance [32]. Investigations of erythroblast GDF15 regulation [14] or caspase related GATA-1 expression [33] can be explored further. Based upon its Title Loaded From File simplicity and reproducibility, the model is being developed for preclinical assessment and comparison with beta thalassemia-related discoveries made in other model systems including biochemical assays, immortalized cell lines, or genetically modified rodents [22]. Since the shRNA in this study does not target gamma-globin mRNA, it should be possible to screen and study fetal hemoglobin augmenting drugs or small molecules for their potential to correct the beta-thalassemia phenotype.Supporting InformationTable S(DOCX)Table Sof insoluble alpha hemoglobin seen in the Western analyses is unknown, but may be related to the absence of macrophage clearance of the apoptotic cells or ubiquitination of the free alphaglobin chains [13]. The accumulation of free alpha-globin in the membranes coincided with apoptosis of the beta-KD cells during the period of maturation when hemoglobin accumulates in donormatched control cells. Despite several similarities between the ex vivo beta-KD model and clinical defects in erythropoiesis reported in vivo for betathalassemia, it must be emphasized that the findings do not completely reflect the beta-thalassemia phenotype. The experi-(DOCX)AcknowledgmentsWe thank the Department of Transfusion Medicine for CD34+ cell Title Loaded From File collection and processing.Author ContributionsConceived and designed the experiments: JLM. Performed the experiments: YTL KSK JFV CB SJN ERM. Analyzed the data: YTL KSK JFV CB SJN ERM AR. Wrote the paper: YTL KSK JLM.A Sy.Additional cell membrane alterations that occur in vivo, microcytosis was not evident [23,24]. Among the enucleated cells, hypochromia was obvious as several cells demonstrated an erythroid ghost-like appearance. However, the low number of enucleated beta-KD cells prevented more formal analyses of those cells. Enucleated cells were reportedly absent from ex vivo cultures of erythroblasts obtained from blood of thalassemia patients [25]. While we were unable to obtain the appropriate clinical samples for direct comparisons to be made between ex vivo cultures of betaKD versus thalassemia-major erythroblasts as part of this study, such comparisons should be made in laboratories where those cells are available. As demonstrated from other areas of biomedical research [26], there exists an urgent need for disease models using primary human cells or tissues. Considerable resources have been utilized in recent decades to develop and explore 11967625 murine models of hemoglobinopathies. Despite their robustness for understanding murine erythropoiesis in vivo, those disease models have not yet led to successful clinical trials for improving the health for patients with thalassemia. Differences between the kinetics of murine and human erythroblast differentiation [27], iron biology [7,28?0], and globin gene regulation [31] confound our ability to interpret the murine data in the context of clinical application. As a result, a considerable gap is developing between basic and clinical research for 1315463 beta-thalassemia. This study was undertaken to characterize the effects of betaglobin chain imbalances upon ex vivo erythroblast differentiation and survival using primary human cells. Our model for knockdown of beta-globin gene expression may also permit examinations of dysfunctional erythroblast heme metabolism or mitochondrial function caused by globin chain imbalance [32]. Investigations of erythroblast GDF15 regulation [14] or caspase related GATA-1 expression [33] can be explored further. Based upon its simplicity and reproducibility, the model is being developed for preclinical assessment and comparison with beta thalassemia-related discoveries made in other model systems including biochemical assays, immortalized cell lines, or genetically modified rodents [22]. Since the shRNA in this study does not target gamma-globin mRNA, it should be possible to screen and study fetal hemoglobin augmenting drugs or small molecules for their potential to correct the beta-thalassemia phenotype.Supporting InformationTable S(DOCX)Table Sof insoluble alpha hemoglobin seen in the Western analyses is unknown, but may be related to the absence of macrophage clearance of the apoptotic cells or ubiquitination of the free alphaglobin chains [13]. The accumulation of free alpha-globin in the membranes coincided with apoptosis of the beta-KD cells during the period of maturation when hemoglobin accumulates in donormatched control cells. Despite several similarities between the ex vivo beta-KD model and clinical defects in erythropoiesis reported in vivo for betathalassemia, it must be emphasized that the findings do not completely reflect the beta-thalassemia phenotype. The experi-(DOCX)AcknowledgmentsWe thank the Department of Transfusion Medicine for CD34+ cell collection and processing.Author ContributionsConceived and designed the experiments: JLM. Performed the experiments: YTL KSK JFV CB SJN ERM. Analyzed the data: YTL KSK JFV CB SJN ERM AR. Wrote the paper: YTL KSK JLM.A Sy.
