Section Editors
Anupam Sachdeva, Shubha R Phadke
- Physiology of HematopoiesisVinod Gunasekaran, Anupam Sachdeva
- Developmental Hemostasis and Physiology of Hemostasis in the Fetus and the NewbornShrimati Shetty
- Granulocytes: Development and PhysiologyTulika Seth
- Developmental Aspects of PlateletsSangeeta Mudaliar
- Fetal AnemiaMeenal Agarwal
- Thalassemia in the Fetus: Prenatal DiagnosisPrajnya Ranganath
- Genetic Counseling for Hematological DisordersRoshan Colah, Khushnooma Italia
- Fetal Hematological Disorders: Future AheadShubha R Phadke
INTRODUCTION
Hematopoiesis is an integral part in the growth process of embryo into fetus and later into an adult. It is essential for survival of fetus and to produce hematopoietic stem cells, which later contributes to hematopoiesis throughout the life. In the fetus, the blood cells are the first among the different cell types to become functionally mature. Erythroid cells are the first among the blood cells to be seen in the conceptus. Throughout the fetal development, hematopoiesis occurs in multiple sites before getting shifted to the final sites (bone marrow and thymus).1 The synthesis occurs in waves beginning in the extraembryonic yolk sac early in the fetal life, later involving arterial cell clusters before migrating to fetal liver and finally seeding the bone marrow. The initial blood cells synthesized have limited progenitor activity, while the definitive adult-type hematopoietic stem cells (HSCs) have multi-lineage differentiation. The hemoglobin produced in early fetal development also differs from that of adult hemoglobin. This chapter discusses these physiological processes in detail.
STEPS IN FETAL HEMATOPOIESIS
The fetal hematopoiesis occurs sequentially in four structures as follows and is illustrated in Flow chart 1.1:
- Extraembryonic yolk sac
- Embryonic arterial cell clusters
- Fetal liver
- Bone marrow.
Yolk Sac Hematopoiesis (Mesoblastic Phase)
Fetal hematopoiesis first starts in the mesoderm of extra embryonic yolk sac (seen as blood islands attached to vascular endothelium).2 These are seen as early as 16 days of embryonic development. The cells initially produced here are primitive erythroid cells, macrophages and megakaryocytes.1 At 4.5 weeks of development, various clonogenic progenitors of these series of cells have been documented. Fetal cardiac activity and blood circulation starts at day 21 of development. Yolk sac hematopoiesis totally disappears after 6–8 weeks of gestation. Thus, this phase is short lived (16 days–8 weeks of gestation) as compared to birds and rodents.2
Embryonic Arterial Cell Clusters
Previously, it was thought that the yolk sac generated stem cells seed fetal liver and bone marrow and is the sole source of hematopoiesis throughout the adult life. But, it has been observed that as the embryo matures, the major arteries in embryo, yolk sac and placenta produce HSCs.
At 27th day, these are first detected attached to aorta, and are seen in increased numbers by 35th day to finally disappear by 40th day.2 Precursors of aorta (splanchnopleura) are intrinsically found to be capable of generating HSCs on 19th day (i.e. prior to onset of blood circulation), thereby confirming the embryonic origin rather than the migratory HSCs from yolk sac.
4Also these cells are exclusively capable of generating B- and T-lymphocytes unlike the yolk sac derived cells. The HSCs do not differentiate further in these sites rather they migrate to fetal liver, bone marrow and thymus for further differentiation.2
In both yolk sac and embryonic origin of HSCs, these arise in proximity to vascular endothelium, thereby leading to a postulation that a common precursor angiohematopoietic cell (hemangioblast) differentiates into hematopoietic as well as vascular endothelial cells. Recently, human placenta in first trimester has been found to be niche which supports the terminal differentiation of primitive erythroid cells along with interactions from macrophages.
Liver Hematopoiesis (Hepatic Phase)
As the blood circulation is established, these HSCs from yolk sac reaches the embryo and seed the fetal liver first from where hematopoiesis occur till the seeding of bone marrow occurs later. Liver is embryologically derived from the endodermal diverticulum of foregut and the mesodermal structure septum transversum (starting from 22nd day of gestation). At 23rd day, first hepatic colonization is said to occur by CD34 negative erythromyeloid cells. A 2nd colonization occurs at 30th day by CD34+ late stage progenitors (derived from embryonic arterial cell clusters), after which liver is able to sustain hematopoiesis (by 32nd day). Embryo–to–fetal hemoglobin switch including the shift in synthesis of ε chains to α and γ chains occur in the liver. However, recent studies have shown that definitive erythropoiesis occurs as a second wave in yolk sac as well.1
Bone Marrow Hematopoiesis (Myeloid Phase)
In bone marrow, hematopoiesis starts from 11th week (10.5 weeks) of gestation. Primary logettes are specialized mesenchymal structures in marrow from where it occurs. The niche supporting these cells comprises of fibrillar material and osteoblasts. Myeloid series are the earliest to differentiate followed by erythroid cells. CD34+ HSCs are however initially absent in the bone marrow and are acquired later which are capable of sustaining hematopoiesis all through the life. At birth, the hematopoiesis is predominant in marrow.3 The fetus contains HbFα2 γ2 (90%) and HbAα2β2 (5–10%).4 Fetal hemoglobin (HbF) has a left-shifted oxygen dissociation curve (high affinity to oxygen), facilitating oxygen delivery to fetus from the placenta.3 Adult hemoglobin (HbA) is first detected in fetal circulation at 13th week.3 But the fetal-to-adult switch in hemoglobin production (HbF → HbA) starts at around 32 weeks of gestation and is completed shortly after birth.1 In circulation, this process is largely complete by 6 months of postnatal life.4 HbA at birth is around 20–30% of the total Hb.3 A normal adult has HbA (97%), HbA2α2δ2 (2.5%) and HbF (0.5%).4
NORMAL BLOOD COUNTS IN FETUS
Over a period of time (from 18th week to 30th week of gestation), parameters including white cell count (4.68 × 109/L–7.71 × 109/L), hemoglobin (11.69–13.64 g%), hematocrit (HCT) and red blood cell (RBC) count progressively increases whereas mean corpuscular volume decreases from 131 fL to 114 fL. Platelet count remains comparable to adult values throughout this time. The platelet counts are also found to increase from 32 weeks of gestation till birth and continues to rise during the first 9 weeks after birth to a level of around 7,50,000/µL. These platelets in neonates are however found to be hyporesponsive to agonists with decreased granule secretion and expression of fibrinogen binding sites for the initial 2–4 weeks of life.5 Differential count shows lymphocyte predominance (80%) throughout this time.6 The time points of significant events in fetal hematopoiesis are summarized in Table 1.1.
REGULATION OF HEMATOPOIESIS
Unlike most other organs, bone marrow has to continuously regenerate hematopoietic elements throughout the life, even when introduced into another host. This capacity of the bone marrow forms the basis for blood donations and administration of myelosuppressive chemotherapy in malignancies. It is also this phenomenon which has formed the basis of hematopoietic stem cell transplantation in the recent years. This continuous renewal occurs in sequential steps of maturation and differentiation from a multipotent stem cell. During progressive steps, this cell gets committed to definite cell lineages. This process occurs in stroma and is regulated by a variety of growth factors and hormones. Hematopoietic stem cells (HSCs) are also regulated by 5signals from neighboring cells during the continuous self-renewal process. This microenvironment is called the niche. HSCs differentiate to wide variety of hematopoietic elements including erythrocytes, platelets, granulocytes (eosinophil, basophils and neutrophils), lymphocytes (T, B and NK cells), monocyte-macrophage system, dendritic cells and mast cells. Any derangements in the niche of hematopoiesis lead to wide range of pathological conditions including anemia, neutropenia, thrombocytopenia, pancytopenia and leukemia.
Regulation of Yolk Sac Hematopoiesis
The visceral endoderm layer of yolk sac regulates mesoblastic phase of hematopoiesis by secreting certain factors including Indian Hedgehog and vascular endothelial growth factor (VEGF). GATA4, a transcription factor is essential for differentiation of visceral endoderm.7 Absence of this factor leads to inability to form blood cell islands in this phase. The primitive erythroid progenitors express receptors for stem cell factor (named c-Kit), transforming growth factor-β, erythropoietin, angiopoietin and VEGF, thereby making these factors regulate hematopoiesis.
Regulation of Embryonic Arterial Cell Clusters
The tissues located ventrally to dorsal aorta are found to support HSCs. This niche supports hematopoiesis through cytokines, soluble factors and physical anchorage. Some of these factors include Flt3 ligand, stem cell factor and interleukin-3 (IL-3).7 Catecholamines also exert influence via sympathetic nerves around arteries located here.
Regulation of Liver Hematopoiesis
The HSCs in fetal liver is supported by a niche consisting of sinusoidal endothelial cells, stromal fibroblasts, macrophages and hepatoblasts. Secretory factors including stem cell factor, IL-6, IL-7, erythropoietin and thrombopoietin secreted by hepatoblasts regulate this phase.7 HSCs adhere to sinusoidal endothelial cells which help in orientation of HSCs and in mitosis. Liver macrophages form a microenvironment called erythroblastic island and help in engulfing the nuclei expelled by the erythroid precursors. A central macrophage is surrounded by a mixed type of cells along with erythroid precursors to form this erythroblastic island, providing a niche for erythropoiesis.
Regulation of Fetal Bone Marrow Hematopoiesis
The long bones develop from mesodermal progenitors initially as cartilaginous bone templates that later get vascularized and form epiphyseal plates. Along with vascular endothelial cells (that form blood vessels), osteoblasts also occupy the bone marrow cavity and play a key role in the establishment of fetal bone marrow niche for HSCs. The process of HSCs homing the marrow and starting hematopoiesis is regulated by chemotactic factors (CXCL12 and its receptor CXCR4 along with other associated molecules).7 Animal studies have shown that mutation of these factors leads to clinical condition with normal liver hematopoiesis but defective homing of bone marrow with HSCs.
Regulation of Neonatal and Juvenile Bone Marrow Hematopoiesis
Like any other organ, bone marrow also needs to adapt to the profound physiological changes that occur after birth. Onset of breathing leading to changes in oxygen content of blood and exposure to external environment leading to immune system activation are the major changes to tackle by the neonatal bone marrow. The exact changes in bone marrow niche are not exactly delineated yet.7 Recognition of increased calcium ions released by the bone matrix is a crucial step. As the ossification process ensues with age, preserving a niche for lifelong hematopoiesis is essential which is regulated by transcription factors.
Regulation of Adult Bone Marrow Hematopoiesis
In an adult bone, HSCs are seen predominantly in metaphyseal regions, but also in diaphyseal and epiphyseal regions. They are localized in two distinct niches in endosteal region (close to bone) and in perivascular region (close to arterioles). HSCs are adherent to osteoblasts which supports hematopoiesis. The role of osteoclasts in hematopoiesis is controversial. The endosteal niche is regulated by various cytokine signaling and cell adhesion molecules. The HSCs in perivascular niche is supported by the surrounded mesenchymal type cells. These mesenchymal cells secrete stem cell factor, CXCL12 thereby regulating hematopoiesis. Megakaryocytes and thrombopoietin are also found to be essential for normal hematopoiesis.7 As aging occurs, hematopoietic cells in the bone marrow are gradually replaced by yellow adipose tissue in selected areas. This adipose tissue has been found to have an inhibitory effect on the HSCs.
STRESS ERYTHROPOIESIS
In the postnatal life, erythropoiesis is usually restricted to bone marrow. But under anemic stress including blood loss, hemolysis (sickle cell anemia, thalassemia), infiltrative conditions (leukemia, myelofibrosis), the erythroid precursors home the extramedullary organs (spleen and liver) and begins erythropoiesis. Erythroblastic islands are formed here. This clinically manifests with splenomegaly and hepatomegaly.
ERYTHROID LINEAGE DIFFERENTIATION
Primitive Erythropoiesis
For a short duration of around 48 hours, the hematopoiesis in yolk sac is predominated by primitive erythroid cells.1 These are large nucleated cells. The differentiated elements of these cells are detected in the circulation even after birth though in a minority. Gastrulation is a developmental step in 6embryo in which a single-layered blastula is reorganized into a three-layered gastrula (having ectoderm, mesoderm and endoderm). The mesoderm gives rise to primitive erythroid cells. Various transcriptional factors (GATA1, EKLF/KLF1, etc.), signaling from the endoderm, TGF-β and Wnt/β catenin signaling pathways regulate initiation and further differentiation.1 Macrophages are the first blood cells to appear in fetal circulation, whereas neutrophils are the last to appear.6 Platelets are first seen at 8–9 weeks of gestation.6 The primitive erythroid cells differ from the definitive erythroid cells (produced later in fetal liver and bone marrow). They possess different globin genes with different O2 carrying capacity and also differ in response to hypoxia, cytokines and various regulatory pathways. These cells synthesize embryonic hemoglobins (having ζ and ε chains). These include Hb Portland (ζ 2γ2), Hb Gower I (ζ 2 ε2) and Hb Gower II(α2ε2).4 These primitive cells are also found to retain the nucleus within circulation till around mid-gestation after which the mature enucleated cells circulate till shortly after birth.1 This is in contrast to the definitive eythroid cells which get enucleated before being released in the circulation. The nucleated primitive cells in circulation undergo maturation after reaching fetal liver. The proerythroblast develops to orthochromatic erythroblast and later into reticulocyte (after enucleation). Macrophages nurse these cells and phagocytose the expelled nuclei. Their lifespans are similar to that of definitive erythroid cells and are destroyed later by spleen.1
Definitive Erythropoiesis
The first definitive erythrocytes are released from the fetal liver. Simultaneously, the hemogloblin synthesis is switched to HbF/HbA containing α, β and γ globin chains. The maturation of HSCs to erythrocytes occurs in serial steps with commitment into specific lineages and maturation occurring at various stages.8 The stages of development erythropoiesis and thrombopoiesis are depicted in Flow chart 1.2.
DIFFERENTIATION OF WHITE BLOOD CELLS
The differentiation of white blood cells from HSCs is depicted in Flow chart 1.3. The various precursors of granulocytes and monocytes formed in the bone marrow during the development process are illustrated in Flow chart 1.4.
CLINICAL SIGNIFICANCE OF FETAL HEMATOPOIESIS
- In β-thalassemia, as a result of decreased or absent β-chain synthesis, HbF levels are persistently increased beyond 6 months of age. These are not due to compensatory rise in HbF, but because of survival advantage of RBCs with HbFα2γ2
- In case of deletion of whole genes encoding β- or δ-globin chains, variable increase in γ chain production occur leading to two different clinical conditions (hereditary persistence of fetal hemoglobin (HPFH) (with no anemia due to sufficient γ chain production) and δβ thalassemia (with moderately severe anemia due to insufficient γ chain production)4
KEY POINTS
- Hematopoiesis occurs in different waves during fetal life (mesoblastic, embryonic arterial cell clusters, hepatic and myeloid phases)
- In yolk sac, primitive hematopoiesis starts at 16 days and disappears by 6–8 weeks of gestation
- Fetal liver starts definitive hematopoiesis by 32nd day of gestation.
- Bone marrow starts hematopoiesis by 10.5 weeks of gestation and continues throughout the life
- Fetal-to-adult switch in hemoglobin synthesis starts by 32 weeks of gestation and is largely complete by birth
- The fetus contains HbFα2γ2 (90%) and HbAα2β2 (5–10%)
- In circulation, adult hemoglobin (HbA) accounts for 20–30% of total hemoglobin at birth and reaches the adult value of around 97% by 6 months of age.
- Differentiation of HSCs is regulated by various mechanisms including stem cell factor, Indian hedgehog signaling, chemokines (CXCL12 and CXCR4), erythropoietin and thrombopoietin along with the surrounding cellular niche.
REFERENCES
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- Tavian M, Peault B. Embryonic development of the human hematopoietic system. Int J Dev Biol. 2005;49:243–50.
- Finne PH, Halvorsen S. Regulation of Erythropoiesis in the Fetus and Newborn. Arch Dis Childhood. 1972;47:683–7.
- Wood WG, Weatherall DJ. Developmental genetics of the human haemoglobins. Biochem J. 1983;215:1–10.
- Revel-Vilk S. The conundrum of neonatal coagulopathy. Hematology Am Soc Hematol Educ Program. 2012;2012:450–4.
- Forestier F, Daffos F, Catherine N, Renard M, Andreux JP. Developmental Hematopoiesis in Normal Human Fetal Blood. Blood. 1991;77(11):2360–3.
- Al-Drees MA, Yeo JH, Boumelhem BB, Antas VI, Brigden KW, Colonne CK, et al. Making Blood: The Haematopoietic Niche throughout Ontogeny. Stem Cells Int. 2015;2015:571893.
- Dzierzak L, Philipsen S. Erythropoiesis: development and differentiation. Cold Spring Harb Perspect Med. 2013:1;3(4): a011601.