SF3B1 is a core component of splicing machinery. Mutations in SF3B1 are frequently found in myelodysplastic syndromes (MDS), particularly in patients with refractory anemia with ringed sideroblasts (RARS), characterized by isolated anemia. SF3B1 mutations have been implicated in the pathophysiology of RARS; however, the physiological function of SF3B1 in erythropoiesis remains unknown. shRNA-mediated approach was used to knockdown SF3B1 in human CD34+ cells. The effects of SF3B1 knockdown on human erythroid cell differentiation, cell cycle, and apoptosis were assessed by flow cytometry. RNA-seq, qRT-PCR, and western blot analyses were used to define the mechanisms of phenotypes following knockdown of SF3B1. We document that SF3B1 knockdown in human CD34+ cells leads to increased apoptosis and cell cycle arrest of early-stage erythroid cells and generation of abnormally nucleated late-stage erythroblasts. RNA-seq analysis of SF3B1-knockdown erythroid progenitor CFU-E cells revealed altered splicing of an E3 ligase Makorin Ring Finger Protein 1 (MKRN1) and subsequent activation of p53 pathway. Importantly, ectopic expression of MKRN1 rescued SF3B1-knockdown-induced alterations. Decreased expression of genes involved in mitosis/cytokinesis pathway including polo-like kinase 1 (PLK1) was noted in SF3B1-knockdown polychromatic and orthochromatic erythroblasts comparing to control cells. Pharmacologic inhibition of PLK1 also led to generation of abnormally nucleated erythroblasts. These findings enabled us to identify novel roles for SF3B1 in human erythropoiesis and provided new insights into its role in regulating normal erythropoiesis. Furthermore, these findings have implications for improved understanding of ineffective erythropoiesis in MDS patients with SF3B1 mutations.

Erythroblastic island (EBI), composed of a central macrophage surrounded by developing erythroid cells, is a structure found in hematopoietic tissues such as fetal liver and bone marrow. It is the first described hematopoietic niche that predominantly supports erythropoiesis. Although it is well accepted that EBIs and EBI macrophage play important roles during erythropoiesis, the mechanisms by which they support erythropoiesis remain largely unclear due to our inability to identify and isolate EBI macrophages. Earlier efforts to identify surface markers for EBI macrophages have focused on the adhesion molecules which are involved in macrophage's interaction with erythroblasts. These include EMP, Vcam1, CD169, CD163, and αV integrin. Findings from these earlier studies suggested that combination of Vcam1, CD169, and mouse macrophage surface marker F4/80 can be used to define mouse EBI macrophage. We found that not all F4/80⁺Vcam1⁺CD169⁺ macrophages are EBI macrophages. Instead, we discovered that EBI macrophages are characterized by the expression of Epor in both mouse and man. RNA-seq analyses of the newly identified EBI macrophages revealed that EBI macrophages have involved specialized function in supporting erythropoiesis. Our findings provide foundation for future studies. Here we will review current knowledge of EBI macrophages and discuss future perspectives.

The present study aimed to investigate the efficacy of RetroNectin-activated cytokine-induced killer cell (R-CIK) therapy in advanced hepatocellular carcinoma patients as compared with conventional chemotherapy, a comparison that has not yet been thoroughly studied. From January 2010 to October 2013, 74 patients with an initial diagnosis of advanced hepatocelluar carcinoma were enrolled in the study. Patients were assigned to one of two treatment arms: patients in arm 1 (n=37) received R-CIK treatment as the first line therapy, whereas those in arm 2 (n=37) received chemotherapy as the first line treatment. The primary end point measured in this study was median overall survival (mOS). Median progression-free survival time (mPFS) and 1- and 2-year survival rates were recorded as secondary end points. Kaplan-Meier analysis was performed on all mOS and mPFS data, and treatment hazard ratios were established using the Cox proportional hazards model. The 1-year survival rate in treatment arm 1 was 42.47% vs. 24.89% in arm 2 (95% CI, 24.91–59.01% vs. 12.10–40.02%, P=0.066); the 2-year survival rates were 21.24 and 5.53% (95% CI, 4.60–45.86 vs. 0.46–21.06%, P=0.106) in arms 1 and 2, respectively; the mPFS in arm 1 was 4.37 vs. 3.90 (x2=0.182, P=0.670) in arm 2; and the mOS in arm 1 was 14.03 months vs. 9.46 months(x2=4.406, P=0.036) in arm 2. Calculations of univariate analyses of arm 1, R-CIK cycles ≥6, KPS >70, AFP ≤400 ng/ml, and findings of no vascular invasion and no extra-hepatic metastasis were potential predictive factors (P70, AFP ≤400 ng/ml, displayed no evidence of vascular invasion, and no extra-hepatic metastasis appeared to have longer survival times compared with other cohorts in the present study.

The fetal liver (FL) is the key erythropoietic organ during fetal development, but knowledge on human FL erythropoiesis is very limited. In this study, we sorted primary erythroblasts from FL cells and performed RNA sequencing (RNA-seq) analyses. We found that temporal gene expression patterns reflected changes in function during primary human FL terminal erythropoiesis. Notably, the expression of genes enriched in proteolysis and autophagy was up-regulated in orthochromatic erythroblasts (OrthoEs), suggesting the involvement of these pathways in enucleation. We also performed RNA-seq of in vitro cultured erythroblasts derived from FL CD34+ cells. Comparison of transcriptomes between the primary and cultured erythroblasts revealed significant differences, indicating impacts of the culture system on gene expression. Notably, the expression of lipid metabolism-related genes was increased in cultured erythroblasts. We further immortalized erythroid cell lines from FL and cord blood (CB) CD34+ cells (FL-iEry and CB-iEry, respectively). FL-iEry and CB-iEry were immortalized at the proerythroblast stage and can be induced to differentiate into OrthoEs, but their enucleation ability was very low. Comparison of the transcriptomes between OrthoEs with and without enucleation capability revealed the down-regulation of pathways involved in chromatin organization and mitophagy in OrthoEs without enucleation capacity, indicating that defects in chromatin organization and mitophagy contribute to the inability of OrthoEs to enucleate. Additionally, the expression of HBE1, HBZ, and HBG2 was up-regulated in FL-iEry compared with CB-iEry, and such up-regulation was accompanied by down-regulated expression of BCL11A and up-regulated expression of LIN28B and IGF2BP1. Our study provides new insights into human FL erythropoiesis and rich resources for future studies.

Erythropoiesis occurs first in the yolk sac as a transit “primitive” form, then is gradually replaced by the “definitive” form in the fetal liver (FL) during fetal development and in the bone marrow (BM) postnatally. While it is well known that differences exist between primitive and definitive erythropoiesis, the similarities and differences between FL and BM definitive erythropoiesis have not been studied. Here we performed comprehensive comparisons of erythroid progenitors and precursors at all maturational stages sorted from E16.5 FL and adult BM. We found that FL cells at all maturational stages were larger than their BM counterparts. We further found that FL BFU-E cells divided at a faster rate and underwent more cell divisions than BM BFU-E. Transcriptome comparison revealed that genes with increased expression in FL BFU-Es were enriched in cell division. Interestingly, the expression levels of glucocorticoid receptor Nr3c1, Myc and Myc downstream target Ccna2 were significantly higher in FL BFU-Es, indicating the role of the Nr3c1-Myc-Ccna2 axis in the enhanced proliferation/cell division of FL BFU-E cells. At the CFU-E stage, the expression of genes associated with hemoglobin biosynthesis were much higher in FL CFU-Es, indicating more hemoglobin production. During terminal erythropoiesis, overall temporal patterns in gene expression were conserved between the FL and BM. While biological processes related to translation, the tricarboxylic acid cycle and hypoxia response were upregulated in FL erythroblasts, those related to antiviral signal pathway were upregulated in BM erythroblasts. Our findings uncovered previously unrecognized differences between FL and BM definitive erythropoiesis and provide novel insights into erythropoiesis.

Emerging evidence indicates that macrophages play important roles in hematopoiesis in addition to their immune functions. The well-known immune-unrelated functions of macrophages include their roles in hematopoiesis, especially the quality control of hematopoietic stem cells (HSCs) and hematopoietic stem and progenitor cells (HSPCs), the support of erythropoiesis, and the regulation of megakaryopoiesis. Several studies, most using mouse models, have explored the roles of macrophages in hematopoiesis in different organs such as the yolk sac (YS), fetal liver (FL), bone marrow (BM), and spleen (SP). We have recently documented the potential roles and underlying mechanisms of macrophages in myeloproliferative neoplasm (MPN), aplastic anemia (AA), and idiopathic thrombocytopenic purpura (ITP). In this article, we review the origin of macrophages, introduce their roles in regulating HSCs/HSPCs, erythropoiesis, and megakaryopoiesis within four hematopoietic organs, and summarize the recent advances of macrophages in MPN, AA, and ITP. Finally, we outline the unresolved questions that future studies should address to explore in greater depth the role of macrophages in both normal and disordered hematopoiesis.