Embryology, Hematopoiesis

Development

Gastrulation begins with the epiblast, a single epithelial cell layer, differentiating into the 3 embryonic germ layers: ectoderm, mesoderm, and endoderm.[5] Within the first 2 weeks of gestation, the initial wave of primitive hematopoietic and endothelial cell development is triggered by signaling to the extraembryonic, endodermal yolk sac. This wave results primarily in the formation of EryP cells, megakaryocytes, macrophages, and endothelial cells.[6]

EryP cells are larger than their progenitors, retain nuclei, express embryonic globins, and are restricted to the yolk sac. These cells contribute to the formation of blood islands—structures in which centrally located cells differentiate into erythroid and myeloid lineages, while peripheral cells become endothelial cells that organize into vascular channels. These blood islands eventually fuse to form an interconnected vascular network spanning the yolk sac.

Oscillatory plasma flows, driven by the developing heart, carry EryP and other primitive cells through these vascular channels.[7] Once in circulation, EryP cells undergo enucleation in the fetal liver, and macrophages subsequently clear the expelled nuclei.[8] EryP production is transient, occurring only during the early phase of yolk sac vascularization. However, the remaining progenitor cells continue to mature, progressing from proerythroblast to orthochromatic erythroblast to reticulocyte, and persist in the bloodstream until at least birth.[9]

Soon after EryP development, highly proliferative, MPPs known as high proliferative potential colony-forming cells (HPP-CFC) emerge in the yolk sac. These cells initiate the 1st wave of definitive hematopoiesis and give rise to EryD. Sometimes referred to as erythroid/myeloid progenitors, these cells serve as a transitional population between primitive erythropoiesis and hematopoiesis derived from hematopoietic stem cells (HSCs). These progenitors migrate to and colonize the fetal liver, the next definitive hematopoietic site during gestation.

The 2nd wave of definitive hematopoiesis replaces both primitive hematopoiesis and the 1st wave of definitive hematopoiesis. HSCs emerge from hemogenic endothelium located within the ventral wall of the developing aorta, specifically in a region known as the paraaortic splanchnopleure. This structure gives rise to the aorta-gonad-mesonephros (AGM) region, which is the primary intraembryonic site for HSC generation.[10]

By the 7th week of gestation, HSCs migrate to and colonize the fetal liver, where they proliferate and begin to differentiate. At this stage, the liver becomes the dominant site of hematopoiesis. HSCs also transiently colonize the spleen around the 20th week, contributing to erythropoiesis for a brief period. Simultaneously, HSCs begin to seed the bone marrow—a critical transition, as the bone marrow eventually becomes the primary site of erythropoiesis as gestation progresses.

The fetal liver provides a supportive microenvironment that promotes HSC expansion and differentiation through a hierarchical progenitor system. EryD cells arise from these progenitors. EryD cells rapidly surpass EryP in circulation and become the predominant erythroid population.

EryD cells express fetal hemoglobin (HbF), composed of 2 γ-globin chains and 2 α-globin chains, which remains the dominant hemoglobin type throughout most of gestation.[11] Around 32 weeks, a globin switch begins, during which γ-globin expression is downregulated and replaced by β-globin synthesis, resulting in the production of adult hemoglobin (HbA). This transition marks the final stage in erythroid ontogeny and continues after birth.

Ossification of the skeletal system progresses toward the 3rd trimester, and bone marrow begins to develop within the emerging bony cavities. At this stage, the marrow of select bones becomes the primary site of hematopoiesis. Concurrently, erythropoiesis ceases in the liver and spleen as bone marrow assumes dominance in hematopoietic cell production.

In postnatal life, definitive erythropoiesis occurs exclusively in the bone marrow under normal physiologic conditions. In infants, RBC production takes place throughout the spongy and trabecular bone. In adults, however, erythropoiesis becomes restricted to the axial skeleton—specifically the vertebrae, sternum, ribs, and proximal ends of long bones. Within the bone marrow, HSCs generate all mature hematopoietic lineages through a hierarchy of intermediate progenitors, which will be discussed in the following section.[12]

Cellular

Numerous models have been proposed to explain the development of the EryD lineage. HSCs lack lineage-specific markers and, instead, express lineage-negative (lin-), Sca1+, and c-Kit+ (LSK) markers.[13] Three primary groups of cells express these markers: long-term HSCs (mainly CD34+ with rare CD34- cells above this lineage, and Flt3-), short-term HSCs (CD34+ and Flt3+), and multipotent progenitor cells (MPP) (CD34+ Flt3+).[14]

One model suggests that MPPs give rise to common lymphoid (CLP) and myeloid (CMP) progenitors. From CMP, megakaryocytic erythroid progenitors (MEPs) differentiate into erythroid progenitors. In mice, MEPs are typically identified by the marker profile c-Kit+, CD34-, CD71 low, and CD16/32-. In humans, the phenotypic profile of MEPs includes CD45+, GPA-, IL-3R-, CD34+, CD36-, and low CD7. An alternative model debates whether hematopoiesis follows a hierarchical progression through MPP or long-term and short-term HSCs, proposing instead that megakaryocytes and erythrocytes arise directly from HSCs.[15] While other models have been described, this activity will focus on the former for further discussion.

The earliest erythroid progenitors arise from MEPs and can form burst-forming units (BFU-E), large red colonies containing thousands of hemoglobinized cells. Bust-forming units share the same immunohistochemical markers as MEPs, making it difficult to distinguish between the two. These cells respond to erythropoietin (EPO), stem cell factor, interleukins 3 and 6, corticosteroids, and insulin-like growth factor 1. The cells then divide to form colony-forming unit–erythroid (CFU-E) cells, which, at this stage, are committed to erythroid differentiation and depend on EPO for survival. In humans, CFU-E displays the phenotype CD45+, GPA-, IL-3R-, CD34-, CD36+, and CD71 high, while in mice, it is characterized by IL-3R-, c-Kit+, and CD71 high.[16]

The transition from CFU-E to proerythroblast involves the loss of c-Kit and the gain of Ter119 expression. EPO stimulates cell division, promotes the expression of erythroid-specific genes, and prevents apoptosis. Proerythroblasts undergo 3 mitoses, resulting in 2 basophilic, 4 polychromatic, and 8 orthochromatic erythroblasts, which ultimately yield 16 reticulocytes. After each division, the maturing erythroblast decreases in size, its nucleus condenses, and hemoglobin accumulates in the cytoplasm. By the orthochromatophilic stage, the erythroid cell exits the cell cycle, resulting in a condensed nucleus that is polarized to one side of the cytoplasm. The nucleus is extruded, forming the reticulocyte. Reticulocytes express high levels of CD71, which they lose as they mature into fully developed RBCs, at which point they gain CD235a+ expression.[17]

Molecular Level

Induction of HbF is a tightly regulated process dependent on the expression of several genes.[18] Three key genetic loci influencing HbF levels include a region on chromosome 2 within the BCL11A gene, an intergenic area between the HBS1L and MYB genes on chromosome 6, and variants within the β-globin locus on chromosome 11.

Extensive research has focused on the BCL11A gene, which plays a critical role in the switch from HbF to HbA. BCL11A mediates the silencing of γ-globin gene expression by interacting with transcription factors such as SOX6, which binds to chromatin at the proximal γ-globin promoter sites. The erythroid transcription factor KLF1 regulates BCL11A by silencing its expression.[19] Additionally, variants near the MYB gene on chromosome 6 have been shown to influence HbF levels, though the exact mechanism remains unclear.[20] Understanding the genes that control the transition from HbF to HbA is crucial for developing treatments for inherited hemoglobinopathies, as therapies that reduce mutated β-globin and upregulate γ-globin may help alleviate symptoms.

Clinical Significance

Errors in the development of HbA can lead to significant consequences for the newborn infant until adulthood. β-thalassemia is a genetic mutation where β-globin chain synthesis is reduced or absent.[21] Impaired β-globin production decreases hemoglobin and RBC formation, causing anemia. More than 200 mutations have been reported, with the majority arising from point mutations in the functionally essential gene regions for β-globin chain production.

A reduced supply of β-globin chains leads to an excess of α-globin chains, which accumulate and aggregate within the bone marrow. The conglomeration of α-globin chains leads to the premature death of erythroid precursors and ineffective erythropoiesis. Anemia stimulates the production of EPO, and this drive to produce more RBCs results in extramedullary hematopoiesis.[22] β-thalassemia has many different types, with severity ranging from asymptomatic to severely symptomatic.

Sickle cell anemia results from a nonconservative missense mutation, which involves a single nucleotide change in the β-globin gene region.[23] This mutation causes RBCs to adopt a "sickle" shape when deoxygenated. These sickled RBCs, which contain hemoglobin S (HbS), can continuously change their form, leading to their premature destruction intravascularly.[24] In their sickled shape, the RBCs tend to entrap themselves in the capillaries, causing vasoocclusion. Excessive vasoocclusion can result in a sickle cell pain crisis.

In both diseases, hydroxyurea has been shown to improve patients' quality of life.[25] This drug increases the levels of HbF in the bloodstream. Since HbF lacks β-globin chains, it is unaffected by the mutations causing these diseases.