Mammalian erythropoiesis occurs within erythroblastic islands (EBIs), niches where maturing erythroblasts interact closely with a central macrophage. While it is generally accepted that EBI macrophages play an important role in erythropoiesis, thorough investigation of the mechanisms by which they support erythropoiesis is limited largely by inability to identify and isolate the specific macrophage sub-population that constitute the EBI. Early studies utilized immunohistochemistry or immunofluorescence to study EBI morphology and structure, while more recent efforts have used flow cytometry for high-throughput quantitative characterization of EBIs and their central macrophages. However, these approaches based on the expectation that EBI macrophages are a homogeneous population (F4/80+/CD169+/VCAM-1+ for example) provide an incomplete picture and potentially overlook critical information about the nature and biology of the islands and their central macrophages. Here, we present a novel method for analysis of EBI macrophages from hematopoietic tissues of mice and rats using multispectral imaging flow cytometry (IFC), which combines the high-throughput advantage of flow cytometry with the morphological and fluorescence features derived from microscopy. This method provides both quantitative analysis of EBIs, as well as structural and morphological details of the central macrophages and associated cells. Importantly, the images, combined with quantitative software features, can be used to evaluate co-expression of phenotypic markers which is crucial since some antigens used to identify macrophages (e.g., F4/80 and CD11b) can be expressed on non-erythroid cells associated with the islands instead of, or in addition to the central macrophage itself. We have used this method to analyze native EBIs from different hematopoietic tissues and evaluated the expression of several markers that have been previously reported to be expressed on EBI macrophages. We found that VCAM-1, F4/80, and CD169 are expressed heterogeneously by the central macrophages within the EBIs, while CD11b, although abundantly expressed by cells within the islands, is not expressed on the EBI macrophages. Moreover, differences in the phenotype of EBIs in rats compared to mice point to potential functional differences between these species. These data demonstrate the usefulness of IFC in analysis and characterization of EBIs and more importantly in exploring the heterogeneity and plasticity of EBI macrophages.
Terminal erythroid differentiation occurs in the bone marrow, within specialized niches termed erythroblastic islands. These functional units consist of a macrophage surrounded by differentiating erythroblasts and have been described more than five decades ago but their function in the pathophysiology of erythropoiesis has remained unclear until recently. Here we propose that the central macrophage in the erythroblastic island contributes to the pathophysiology of anemia of inflammation. After introducing erythropoiesis and the interactions between the erythroblasts and the central macrophage within the erythroblastic islands, we will discuss the immunophenotypic characterization of this specific subpopulation of macrophages. We will then integrate these concepts into the currently known pathophysiological drivers of anemia of inflammation and address the role of the central macrophage in this disorder. Finally, as a means of furthering our understanding of the various concepts, we will discuss the differences between murine and rat models with regards to developmental and stress erythropoiesis in an attempt to define a model system representative of human pathophysiology.
Purpose of review The erythroid progenitors BFU-E (burst-forming unit-erythroid) and CFU-E (colony-forming unit-erythroid) have a critical role in erythropoiesis. They represent a heterogeneous and poorly characterized population of cells with modifiable self-renewal, proliferation and differentiation capabilities. This review focuses on the current state of erythroid progenitor biology with regard to immunophenotypic identification and regulatory programs; in addition, we will discuss the therapeutic implications of using these erythroid progenitors as pharmacologic targets. Recent findings Erythroid progenitors are classically defined by the appearance of morphologically defined colonies in semisolid cultures. However, these prior systems preclude a more thorough understanding of the composite nature of progenitor populations. Recent studies employing novel flow cytometric and cell-based assays have helped to redefine hematopoiesis, and suggest that erythroid progenitors may arise from different levels of the hematopoietic tree. Moreover, the identification of cell surface marker patterns in human BFU-E and CFU-E enhance our ability to perform downstream functional and molecular analyses at the population and single cell level. Advances in these techniques have already revealed novel subpopulations with increased self-renewing capacity, roles for erythroid progenitors in globin gene expression, and insights into pharmacologic mechanisms of glucocorticoids and pomalidomide. Summary Immunophenotypic and molecular characterization resolves the diversity of erythroid progenitors and may ultimately lead to the ability to target these progenitors to ameliorate diseases of dyserythropoiesis.
Ribosomopathies are a class of disorders caused by defects in the structure or function of the ribosome and characterized by tissue-specific abnormalities. Diamond Blackfan anemia (DBA) arises from different mutations, predominantly in genes encoding ribosomal proteins (RPs). Apart from the anemia, skeletal defects are among the most common anomalies observed in patients with DBA, but they are virtually restricted to radial ray and other upper limb defects. What leads to these site-specific skeletal defects in DBA remains a mystery. Using a novel mouse model for RP haploinsufficiency, we observed specific, differential defects of the limbs. Using complementary in vitro and in vivo approaches, we demonstrate that reduced WNT signaling and subsequent increased β-catenin degradation in concert with increased expression of p53 contribute to mesenchymal lineage failure. We observed differential defects in the proliferation and differentiation of mesenchymal stem cells (MSCs) from the forelimb versus the hind limbs of the RP haploinsufficient mice that persisted after birth and were partially rescued by allelic reduction of Trp53. These defects are associated with a global decrease in protein translation in RP haploinsufficient MSCs, with the effect more pronounced in cells isolated from the forelimbs. Together these results demonstrate translational differences inherent to the MSC, explaining the site-specific skeletal defects observed in DBA.
Erythroblastic islands (EBIs) are a hallmark of mammalian erythropoiesis consisting of a central macrophage surrounded by and interacting closely with maturing erythroblasts. While it is generally accepted that the island macrophages play an important role in erythropoiesis, the inability to identify and isolate this macrophage subpopulation has limited our understanding of their functional involvement. Previous studies have relied on immunohistochemistry/immunofluorescence in situ or in vitro. More recently, flow cytometry was used to characterize EBI formation and the immunophenotype of the central macrophages in murine erythroblastic islands. These approaches provide either morphological/structural information or high-throughput quantification, but not both, and often carry the expectation that all EBI macrophages have similar phenotype (F4/80+/CD169+/VCAM1+ for example), and thus potentially overlook critical information about the nature and biology of the islands and the central macrophages. We have developed a novel method for analysis and characterization of EBI macrophages from hematopoietic tissues using multispectral imaging flow cytometry, which combines the high-throughput advantage of flow cytometry with the morphology and fluorescence details obtained from microscopy. This method allows automated, non-biased evaluation of the EBIs recovered from a sample, their number, mean size, as well as structural and morphological details of the central macrophages and associated erythroblasts. Most importantly, the images, combined with the fluorescence similarity feature, enables the evaluation of co-expression of any phenotypic markers that may be used to identify the macrophages which is crucial since some antigens used to identify macrophages (e.g. CD45, CD11b) may also be expressed on non-erythroid cells associated with the islands instead of, or in addition to, the central macrophage itself. We used this method to confirm the expression of various markers previously reported to be expressed on the erythroblastic island macrophages by flow, including CD11b, VCAM1, F4/80, CD169, and CD163, in mouse, rat, and human bone marrow. Indeed, while a large number of studies have focused on murine erythropoiesis, the identity and role of the EBIs in other species is much less known. We confirmed expression of CD169 and VCAM1 on the F4/80+ central macrophages of murine EBIs and also identified a population of VCAM+/F4/80- central cells associated with developing erythroblasts. CD11b is abundantly expressed by non-erythroid, non-macrophage cells associated with the islands, but is not expressed significantly on the central macrophages (Figure 1). CD163, a marker of EBI macrophages in rat and human, was not detected in the murine EBIs by imaging flow cytometry, but this may be due to limitation of the antibodies tested. In contrast, anti-CD163 stained well rat and human EBI macrophages but CD11b or VCAM1 were not detected in EBIs from rat and human bone marrow respectively, raising the question of a species-specificity regarding the macrophage heterogeneity and satellite cells present within erythroblastic islands. In summary, the data presented herein demonstrate the effectiveness of this method for the analysis and characterization of EBIs and establish a new tool for future investigations of EBIs and their central macrophages in the nurturing of erythropoiesis. Figure 1 Representative image of an erythroblastic island harvested from murine bone marrow stained with F4/80-AF488 (green), CD11b-PE (blue), and CD71-BV421 (red) and analyzed by imaging flow cytometry. Figure 1. Representative image of an erythroblastic island harvested from murine bone marrow stained with F4/80-AF488 (green), CD11b-PE (blue), and CD71-BV421 (red) and analyzed by imaging flow cytometry. Disclosures No relevant conflicts of interest to declare.
DBA is an inherited bone marrow failure syndrome characterized by pure red cell aplasia, congenital physical anomalies, and cancer predisposition, in particular an increased incidence of osteogenic sarcoma (O:E 42.44). In order to investigate the mechanism of defective bone development and ultimately oncogenesis, we used an in vitro model consisting of mouse embryonic stem cells gene trapped for the Ribosomal Protein 19 gene (Rps19) and developed an in vivo transgenic conditional mouse model with floxed Rps19 alleles and a bone-specific expressing Col1a1-CreER. Using a 23-day stepwise osteoblast differentiation model, haploinsufficient Rps19+/- mouse ES cells did not show differences in the ability to develop into mesodermal progenitors based on qPCR analysis of mesodermal transcription factors, but displayed a decreased ability to produce mature osteoblasts based upon decreased alizarin red staining. In addition, higher levels of the Trp53 protein were found in Rps19+/- mouse ES cells at both the pluripotent and mesodermal stages of differentiation. Further, Rps19+/- mouse ES cells presented with aberrant WNT signaling, as represented by decreased levels of β-catenin early in osteoblast differentiation and rising to higher levels towards late osteoblast differentiation. We assessed the hypothesis of WNT signaling involvement in lineage specification and found that Rps19+/- mouse ES cell-derived osteoblasts show decreased levels of Runx2 and increased levels of Sox9. In the transgenic mouse model, tamoxifen-induced recombination of Rps19 at E15-17 did not show gross morphologic skeletal defects at E20.5 for Rps19fl/+ embryos, although Rps19fl/fl embryos displayed severe skeletal defects through alizarin red and alcian blue staining. In conclusion, haploinsufficient levels of Rps19 found in mice contribute to improper skeletal development and probably reflect the clinical scenario. We plan to utilize these in vitro and in vivo models to provide clues into the mechanism of ribosomal protein haploinsufficiency in tumor development, which may suggest surveillance and treatment strategies. Citation Format: Jimmy Hom, Brian Dulmovits, Luanne Peters, Adrianna Vlachos, Jeffrey M. Lipton, Lionel Blanc. Modeling bone development and cancer predisposition in Diamond Blackfan anemia (DBA) [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B41.
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