Biology Of Erythropoiesis, Erythroid Differentiation, And Maturation

Phenotypic and Functional Properties of Erythroid Progenitor Cells

These cells are functionally situated between the multipotent stem cell and the morphologically distinguishable erythroid precursor cells ( Chapter 9 ). This compartment contains a spectrum of cells with a parent-to-progeny relationship, all committed to erythroid differentiation. A complete understanding of how erythroid commitment is achieved at the biochemical or molecular level is finally starting to emerge in all its complexity (see intrinsic control of erythropoiesis) (most of the references published before 2016 are available in Papayannopoulou and Migliaccio ). Although all erythroid progenitor cells share the irreversible commitment to express the erythroid phenotype, the properties of these cells progressively diverge as the cells become separated by several divisions. Over time, increases in the sophistication of the technologies used to determine these properties have also increased the precision with which we define these cells.

Erythroid progenitor cells are sparse and difficult to isolate in sufficient purity and numbers for study. For these reasons, the existence and characteristics of these cells were first inferred by functional assays based on their ability to generate hemoglobinized progeny in vitro in clonal erythroid cultures. Two classes of progenitors have been identified using this approach. The first, more primitive class consists of the burst-forming unit-erythroid (BFU-E), named for the ability of BFU-E to give rise to multiclustered colonies (erythroid bursts) of hemoglobin-containing cells. BFU-E represent the earliest progenitors committed exclusively to erythroid differentiation and a quiescent reserve, with only 10% to 20% in cycle at any given time. However, once stimulated to proliferate in the presence of appropriate cytokines, BFU-Es demonstrate a significant proliferative capacity in vitro, giving rise to colonies of 30,000 to 40,000 cells, which become fully hemoglobinized after 2 to 4 weeks, with a peak incidence at 14 to 16 days. They have a limited self-renewal capacity; at least a subset of BFU-E is capable of generating secondary colonies upon replating. In contrast to this class of progenitor cells, a second, more differentiated class of progenitors consists of the colony-forming unit–erythroid (CFU-E). Most (60% to 80%) of these progenitors already are in cycle and thus proliferate immediately after initiation of culture, forming erythroid colonies within 7 days. Because CFU-E are more differentiated than BFU-E, they require fewer divisions to generate colonies of hemoglobinized cells, and the colonies are small (8 to 64 cells per colony).

Although BFU-E and CFU-E appear distinct from each other, in reality progenitor cells constitute a continuum, with graded changes in their properties. Only progenitor cells at both ends of the differentiation spectrum have distinct properties. Perhaps the earliest cell with the potential to generate hemoglobinized progeny is an oligopotent progenitor, which is capable of giving rise to mature cells of at least one other lineage (granulocytic, macrophage, or megakaryocytic) in addition to the erythroid. This progenitor, a multilineage colony-forming unit (CFU) called a colony-forming unit–granulocyte , erythrocyte, macrophage, megakaryocyte (CFU-GEMM) or common myeloid progenitor , and the most primitive BFU-E have physical and functional properties that are shared by both pluripotent stem cells and progenitor cells committed to non-erythroid lineages. These properties include high proliferative potential, low cycling rate, response to a combination of cytokines, and presence of specific surface antigens or surface receptors. In contrast, the most differentiated CFU-E have many similarities with erythroid precursor cells and have little in common with primitive BFU-E. Their proliferative potential is limited, they cannot self-renew, they lack the cell surface antigens common to all early progenitors, and they are exquisitely sensitive to erythropoietin (EPO).

Although clonal erythroid cultures are indispensable for the study of erythroid progenitors, they do not faithfully reproduce the in vivo kinetics of red cell differentiation/maturation, and many maturing cells have a megaloblastic appearance and lyse before they reach the end stage of red cell development. In vivo, erythropoiesis probably occurs faster than predicted from culture data. For example, studies in dogs with cyclic hematopoiesis, a genetic stem cell defect leading to pulses of hematopoiesis ( Chapter 30 ), provide evidence that BFU-E mature to CFU-E over 2 to 3 days in vivo, although this process may require 5 to 6 days in canine marrow cultures.

BFU-E and their immediate progeny (but not CFU-E) are motile cells found in significant numbers in peripheral blood. As with BFU-E, the ability of stem cells and progenitor cells to circulate is physiologically important for the redistribution of marrow cells in cases of local damage to the microenvironment and for reconstitution of hematopoiesis after transplantation. The spectrum of BFU-E in circulation probably is narrower (consisting mostly of early, quiescent BFU-E) than that of BFU-E in the bone marrow; otherwise, their properties are similar to those of marrow BFU-E. The number of circulating BFU-E (along with other progenitors and stem cells) can increase to significant levels after cytokine/chemokine treatments and after chemotherapy, a finding that has been exploited for transplantation purposes. At present, mononuclear cells contained in the blood from subjects mobilized with granulocyte colony-stimulating factor (G-CSF) are routinely used as a source of stem/progenitor cells for autologous and allergenic transplantation, alone or in combination with AMD3100, an inhibitor of CXCR4, the receptors expressed on the progenitor cells that by binding with SDF1 (also known as CXCL12) produced by stromal cells, retain the cells in the bone marrow ( Chapter 16 ). Apart from SDF1 another important pathway for retaining stem/progenitor cells in BM is represented by VLA-4 integrins (α4β1). These pathways are independently regulated, but they work in concert.

In addition to forming colonies in semisolid medium, hematopoietic progenitors from different sources can generate erythroid cells in liquid culture. Liquid cultures do not allow progenitor cell enumeration but may generate more differentiated cells per progenitor cell than occurs in semisolid cultures. This culture system is often used for modeling erythroid disorders and in theory may generate numbers of erythroid cells equivalent to 1 unit of blood from discarded stem cell sources (cord blood and leukoreduced products of blood donations), which has led to the belief that red blood cells (RBCs) generated ex vivo may one day be used for transfusion therapies.

The hematopoietic compartments can also be defined on the basis of their antigenic profiling based on the expression on the plasma membrane of specific proteins recognized by monoclonal antibodies. These studies have first provided a robust definition of the progenitor cells present in bone marrow of normal mice where the prospectively isolated cells may be functionally tested not only in vitro but also in vivo.

The best representative antigen expressed by human BFU-E is the CD34 antigen, which has been successfully exploited for isolation of BFU-E and other progenitors. CD34 is a highly O -glycosylated cell surface glycoprotein expressed by all hematopoietic progenitors and vascular endothelial cells that may serve as a bumper that prevents close contacts among these cells. Additional clinically important antigens expressed by human erythroid progenitors are the histocompatibility antigens. Like other hematopoietic progenitors, BFU-E display human leukocyte antigen (HLA) class I (A, B, C) and class II (DP, DQ, DR) antigens on their surface. Class II antigens (especially the products of the DR locus), in contrast to class I, are variably expressed by BFU-E. Furthermore, use of antibodies or conjugated ligands determined that, as most of the other immature hematopoietic progenitor cells, BFU-E display receptors for KIT ligand (KL, also known as stem cell factor, SCF), EPO, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin (IL)-3 (the alpha and common beta subunit for both), gp130, the signaling subunit of the IL-6 receptor, and IL-11. They share with late colony-forming unit–megakaryocyte (CFU-Mk) progenitors the expression of the thrombopoietin (TPO) receptor (c-Mpl or TPO-R) and glycoprotein Iib/IIIa (CD41), an antigen previously thought to be restricted to megakaryocytes, which marks the divergence between definitive hematopoiesis and endothelial cells during development. However, the majority of BFU-E, in contrast to myeloid progenitors, do not express the restricted hematopoietic phosphatase CD45R and aldehyde dehydrogenase activity, an enzyme lost in humans during the transition from CMP to MEP.

As BFU-E mature to the CFU-E stage, they begin to express surface proteins characteristic of erythroblasts, the morphologically recognizable erythroid cells. For example, CFU-E expresses Rh antigens and the erythroid-specific sialoglycoprotein glycophorin A. Blood group antigens of the ABH Ii type are detectable in a subset of CFU-E. In contrast, CD34 molecules, class II antigens, and certain growth factor receptors (i.e., IL-3R, KIT) are greatly diminished or virtually absent at the CFU-E stage. Conversely, the EPO-receptor is greatly expressed at the CFU-E stage but barely detectable on BFU-E. Thus, CFU-E, in contrast to BFU-E, cannot survive in vitro even for a few hours in the absence of EPO.

In conclusion, the Weissman laboratory has defined the phenotype of human erythroid progenitors as lineage ⁻ , CD34 ⁺ , CD38 ⁺ , IL-3 receptor α ⁻ , and CD34RA ⁻ for MEP and Lineage ⁻ , CD34 ⁺ , CD38 ⁺ , IL-3 receptor α ⁻ , CD34RA ⁻ , CD71 ^intermediate , and CD105 ⁺ for erythroid restricted progenitor cells while other investigators have defined the phenotype for human BFU-E as CD45 ⁺ , GPA ⁻ , IL-3 receptor ⁻ , CD34 ⁺ , CD36 ⁻ , and CD71 ^low and that for CFU-E as CD45 ⁺ ,GPA ⁻ , IL-3R ⁻ , CD36 ⁺ , and CD71 ^high .

More recent analyses of single cell transcriptome profiling has identified significant levels of variability even in cells purified at homogeneity on the basis of rigorous antigenic criteria. This analysis may lead to the identification of alternative differentiation routes that may play important roles under pathological or stress conditions. These studies are still too preliminary and further information should shed more light on these issues and may require the development of specific cell culture conditions.

Plasma Membrane and Cytoskeleton Reorganization

The shape and deformability of red cells are determined by the appropriate assembly of their membrane proteins with the cytoplasmic cytoskeleton ( Chapter 48 ). Red cells survive shear forces in the microvasculature because of two transmembrane complexes, ankyrin and 4.1, embedded in the lipid bilayer and attached to the cytoskeleton, ensuring its flexibility. The extracellular part of these complexes contains the clinically relevant blood group antigens determined by genetic polymorphisms in proteins of these complexes. The intracellular parts contain the docking sites for actin filaments, interconnected with (α1β1) ₂ tetramers forming a supporting network below the lipid bilayer of the plasma membrane. Formation of this network is facilitated by the actin-motor protein non-muscle myosin IIA encoded by the MYH9 gene. A recently recognized player required for actin assembly in red cells, Rac GTPase, has been identified.

Most membrane cytoskeletal proteins (spectrin, glycophorin, band 3, band 4.1, and ankyrin) accumulate after the CFU-E stage (i.e., within the precursor cell compartment). Specifically, expression of membrane glycoproteins such as band 3 and band 4.1 is greatly enhanced at the later stages of erythroid maturation. Likewise, the quantity of polylactosaminoglycan, a specific carbohydrate chain that carries blood group ABH and Ii antigenic determinants, is much higher in mature erythrocytes than in erythroblasts ( Chapter 111 ). Whereas a linear, virtually unbranched polylactosamine structure is present in fetal and newborn erythroid cells (reflected by i antigenic reactivity), a branched polylactosaminyl structure is present in adult erythroblasts (reflected by I antigenic reactivity), and branching increases further as maturation progresses.

Glycophorins, especially glycophorin A, are expressed fully at the CFU-E or proerythroblast level just before expression of globin, and few changes occur during maturation. In contrast, the membrane glycoproteins p105 and p95 decline during the later stages of maturation, and yet other membrane glycoproteins, such as vimentin (an intermediate filament protein), are totally lost. Loss of vimentin expression at the late erythroblastic stages most likely facilitates enucleation.

In addition to quantitative changes that occur during maturation, gradual switches in subunit composition of some cytoskeletal proteins occur. For example, exclusively erythroid subunits of α- and β-spectrin are displayed only in end-stage cells. Likewise, multiple transcripts of ankyrin or protein 4.1 have been identified, and the ratios of these transcripts change during maturation. Initial expression of many of these membrane components likely begins at the progenitor cell level. However, in these cells, final assembly may be discouraged because of the higher turnover of these proteins, which minimizes mutual interactions, or because of asynchrony in protein synthesis. Prevention of cytoskeletal assembly at these early stages may secure more membrane fluidity and cell motility needed during this proliferative phase of differentiation. Because molecular probes for many of the red cell cytoskeletal components have been developed, detailed information about the transcription and processing of most of these proteins is beginning to emerge. For example, band 3, the major anion transport protein of human erythrocytes, is a key component of a multicomplex that also contains protein 4.2. Appropriate display of this protein complex on the cell membrane is dependent on critical interactions established between newly synthesized band 3 and protein 4.2 already at the proerythroblast stage.

Expression of the majority of genes encoding cytoskeletal components is not restricted to red cells. Dissecting hematopoietic from non-hematopoietic consequences of abnormalities in these genes has been difficult, but the development of mouse models that mimic defects found in human diseases has been helpful in this respect. It has been presently identified that the erythroid specificity of the expression of these proteins is achieved through alternative splicing of the corresponding messenger RNA (mRNA) mediated by spliceosomes the expression of which increases with the progression of maturation.

Mitophagy and Ribosome and Mitochondria Clearance

The mature red cells do not contain mitochondria, ribosomes, and other cellular elements such as the spindle motors. These structures are eliminated by a specialized process of autophagy-defined mitophagy that begins at the pro-erythroblast levels (see Fig. 27.2 ) and involves clustering of the mitochondria pool at one pole of the cells where they start to display crests with morphological features predictive of degeneration. This region of the cell also contains the rough endoplasmic reticulum. These alterations are probably determined by increased reactive oxygen species (ROS) levels due to iron accumulation ( Chapter 36 ). Mitochondria, and the surrounding rough endoplasmic reticulum, are then embedded into autophagic vesicle where they are posed for degradation. This process requires the proteins NIX and FOXA and is completed at the reticulocyte level ( Figs. 27.1 and 27.2 ). As a consequence of mitophagy, the energy metabolism of erythroid cells becomes exquisitely dependent on the anaerobic pathway. Mitochondria are retained by RBCs in the blood of patients with sickle cell anemia and are thought to be responsible for increased oxygen consumption rates and ROS generation that mediate the high level of hemolysis in these patients.