Hematopoiesis

Hematopoiesis is the formation of blood cells. It starts during embryonic development and continues throughout adulthood to produce and replenish the blood cellular components. Hematopoiesis either medullary (occurred in the bone mar­row) or extramedullary (other than bone marrow) depending on the age of the individuals.

4.2.1 Pluripotential Hematopoietic Stem Cells (PHSC)

These cells are characterized by their long self-renewal capacity and pluripotency. PHSC have the capability to undergo mitotic cell divisions and a portion of these cells remain undifferentiated and maintain a constant pool of PHSC throughout life whereas another portion of PHSC is programmed for differentiation to produce specialized cells. The term pluripotency denotes the ability to differentiate into the cells of a particular lineage. The predominant source of PHSC is the bone marrow (1 in every 100,000 cells in the marrow are PHSC), but they can mobilize the circulation and a substantial number of PHSC are available in peripheral blood. Beside these, PHSC are found in umbilical cord blood, fetal hematopoietic system (yolk sac and fetal liver) and embryonic stem cells and embryonic germ cells. PHSCs migrate to the bone marrow just before birth where they permanently reside throughout life. In human, long bones are the primary site for medullary hematopoiesis up to 20 years of age. Later, the medullary hematopoiesis occurs at flat bones like sternum and vertebrae.

The hematopoiesis can be broadly classified into two categories based on their time of occurrence and the nature of the precursors and progenitor cells. The primitive wave of erythropoiesis mostly seen during embryonic/fetal life in fetal hematopoietic systems characterized by generation of large, nucleated erythrocytes including macrophages and megakaryocytes. The primitive wave is transient and the erythroid progenitors thus produced are neither pluripotent nor having self-renewal property. In contrast, definitive wave is characterized by the development of all blood cell lineages from hematopoietic stem cells occurred during later stage of development. However, there is a transient definitive wave of hematopoiesis that occurs in most of the species produces erythroid-myeloid progenitors (EMPs).

4.2.2 Development of Pluripotential Hematopoietic Stem Cells

All the blood cells are derived from the mesodermal germ layer. The dorsal mesoderm gives rise to somites and noto­chord, whereas the ventral mesoderm is specified for blood and vasculature. The exact mechanism behind the differenti­ation of the mesodermal germ layer into hematopoietic stem cells is still awaiting clarification. In humans, hematopoiesis starts around day 17 of embryonic life in the yolk sac. During the course of prenatal development, the site of hematopoiesis varies considerably. The first hepatic colonization of hematopoietic site occurs around day 23 which then migrates to arterial colonization (day 27), second hepatic colonization (day 30), and finally bone marrow colonization around the 11th week of prenatal development.

4.2.3 Development of Progenitor Cells

The first primitive wave of fetal erythropoiesis occurred in the blood island of extra-embryonic yolk sac from a cluster of early endothelial cells (hemangioblast) gives rise to a large nucleated erythroid progenitor along with macrophages and rare megakaryocyte progenitors.

A second definitive wave of hematopoiesis occurs in the fetal liver from long-term hematopoietic stem cells (LT-HSC) and generates more complex functionally competent adult­like blood cells. This wave also favors the third wave of hematopoiesis. LT-HSC are having self-renewal properties and are differentiated into short-term hematopoietic stem cells (ST-HSC), which undergoes further differentiation into common myeloid progenitors (CMP) or colony forming unit granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), and common lymphoid progenitor (CLP). CMP generates megakaryocyte, erythroid, granulocytes, and macrophages progenitors and CLP give rise to B and T lymphocytes.

The third wave of hematopoiesis occurs in the aorta-gonad- mesonephros (AGM) region of the developing embryo from autonomously generated first adult PHSCs, giving rise to the permanent adult hematopoietic system. The stages are almost similar with second wave of hematopoiesis.

4.2.4 Erythropoiesis

The genesis of erythrocytes from PHScs through series of proliferation and differentiation is called erythropoiesis. The erythropoiesis is aimed either to replenish old and damaged RBCs (basal erythropoiesis) or to cope up the hypoxia due to blood loss or anemia (stress-induced erythropoiesis).

4.2.4.1 Formation of Erythroid Progenitor Cells and Erythroid Precursor Cells

The earliest erythroid progenitor developed from CMP is burst forming unit-erythroid (BFU-E) followed by colony forming units-erythroid (CFU-Es). These erythroid progeni­tor cells undergo differentiation to form erythroid precursor cells termed as proerythroblast/rubriblast/pronormoblast; however, rubriblast is used as a veterinary terminology. These rubriblasts are characterized by basophilic cytoplasm along with centrally located nuclei with deeply stained chro­matin granules. The rubriblasts then undergo a series of differentiation characterized by decrease in cell size with gradual increase in hemoglobin content followed by conden­sation of nuclear chromatin.

4.2.4.2 Terminal Erythropoiesis: Enucleation and Organelle Clearance

Maturation of committed erythroid precursors to form reticulocytes is called terminal erythropoiesis. The terminal erythropoiesis involves some morphological alteration such as reduction in cell size, chromatin condensation, production of hemoglobin, and finally enucleation and elimination of cytosolic organelle such as Golgi apparatus, endoplasmic reticulum, mitochondria, and ribosomes. In early mammalian embryo, enucleation occurs at the fetal liver whereas from mid gestation and during adulthood the principal site of enucleation is the bone marrow. The erythroblastic island of the bone marrow consists of a central macrophage surrounded by erythroblasts and the interaction between mac­rophage and erythroblasts is essentially required for differen­tiation of erythroblasts.

The preparatory phages prior to enucleation include arrest in the cell cycle, chromatin condensation, and nuclear polari­zation. The enucleation process is facilitated by rearrange­ment of specific surface antigens that make erythroid cells prone to macrophage engulfment.

The removal of cell organelle is facilitated by the process of autophagy, characterized by the formation of the autophagosome, its fusion with the lysosome to form phagolysosome, and finally the degradation of organelles by hydrolytic enzymes.

4.2.4.3 Factors Affecting Erythropoiesis

4.2.4.3.1 Tissue Oxygenation: Role of Erythropoietin

Tissue oxygenation is the primary stimulus for erythropoie­sis.

EPO acts through EPO receptor (EPO-R) expressed pri­marily on erythroid progenitor cells like BFU-E and colony forming units-erythroid (CFU-E) though their number varies on the basis of differentiation stages. The signal transduction pathway after activation of EPO-R increases calcium influx inside the cell which in turn regulates expression of proto­oncogenes, phosphorylation of transcriptional factors, or acti­vation of calcineurin and ultimately favors the growth, sur­vival, and differentiation of erythroid committed progenitor cells for the production of proerythroblasts/rubriblasts or by decreasing the rate of apoptosis.

4.2.4.3.2 Nutrition: Iron, Vitamin-B₁₂ and Folic Acid

The nutritional status of the animals, particularly, iron, Vita- min-B₁₂, and folic acid are essentially required for erythro­poiesis, and thus the deficiency of these factors leads to nutritional anemias.

Iron forms the nucleus of the iron-porphyrin heme ring and is required for hemoglobin synthesis. Iron deficiency leads to insufficient hemoglobin formation and microcytic erythrocytes (small size).

Two vitamins, namely vit-B₁₂ and folic acid, are required for the maturation of RBC.

Intrinsic factor (IF), a glycoprotein released from parietal cells, helps in absorption of vit-B₁2 by protecting it from gastric digestion. Lack of intrinsic factor causes loss of vit-B₁2 by the action of digestive enzymes and failure of its absorption.

Vitamin-C helps in iron absorption either by preventing the formation of insoluble and unabsorbable iron compounds and/or by the reduction of ferric to ferrous iron required for the iron uptake into the mucosal cells.

4.2.5 Leukopoiesis

Leukopoiesis is the development of leukocytes. PHSCs are differentiated into common myeloid progenitors (CMP) or colony forming unit granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), and common lymphoid pro­genitor (CLP). CMP generates megakaryocyte, erythroid, granulocytes, and macrophages progenitors and CLP give rise to B and T lymphocytes. CFU-GEMM differentiated into granulocyte-monocyte progenitor (GMP) cells which ultimately give rise to granulocytes or monocytes. CFU eosinophils (CFU-Eo) and basophils (CFU-Ba) differentiated to form eosinophils and basophils.

4.2.5.1 Granulopoiesis and Monocytopoiesis

The granulopoiesis is the development of granulocytes and monocytopoiesis is the development of monocytes. Myelo­blast is the first granulocyte precursor which is differentiated into promyelocytes. Up to promyelocyte stage, the granulo­cyte and monocyte cell lines are similar then the cell lines develop cell lineage-specific granules and form neutrophil myelocyte, eosinophil myelocyte, and basophil myelocyte. The myelocytes then undergo differentiation to form neutro­phil metamyelocyte and eosinophil metamyelocyte. Poly­morphonuclear basophils are developed from basophil myelocyte. However, neutrophil and eosinophil myelocytes undergo another differentiation step called neutrophil and eosinophil meta-myelocytes. The neutrophils have one addi­tional stage of development called “band” neutrophil metamyelocyte before the final stage, i.e., polymorphonu­clear neutrophils. The granules are developed successively during the course of development. The nucleated granules are developed during early pro-myelocyte stage. Azurophil granules are developed during late promyelocyte stage and specific granules are formed during myelocyte stage. The granulocyte precursors can be divided into two pools. The proliferation pool consists of the cells that can be divided into myeloblasts, promyelocytes, and myelocytes. The metamyelocytes and band cells are unable to be divided and categorized under maturation pool. The storage pool is the subdivision of maturation pool which stores mature neutrophils. The storage pool is large in dogs but small in ruminants.

The first monocyte precursor cells developed from GMP are monoblasts which undergo differentiation to form promonocytes and monocytes. In contrast to granulocytes, monocytes don’t have any storage pool, but they enter venous sinusoids and migrate to the tissue where they are differentiated into macrophages.

4.2.5.2 Development of Lymphocytes

The development of lymphocytes occurs in the central/pri- mary lymphoid organs (bone marrow or fetal liver) and thymus for B and T lymphocytes, respectively. The lymphocytes produced in the primary lymphoid organs are then migrated to peripheral lymphoid organs/secondary lym­phoid organs where they interact with the antigens. In adults, the development of T cells in the thymus is decreased, but a continual supply is maintained through division of mature T cells in the peripheral lymphoid organs. In contrast, B cells are produced continuously from the bone marrow in adults. The common lymphoid progenitor (CLP) differentiated into pro-B and pro-T cells which give rise to B and T lymphocytes, respectively.

The antigenic specificity of B and T cells is marked by the immunoglobulins and T cell receptor (TCR), respectively. The differentiation of lymphocytes therefore required genetic programming to express immunoglobulin or TCR, respec­tively, for B and T lymphocytes. The genes responsible for antigen receptors in B cells (immunoglobulin) and T cells (TCR) undergo specific arrangement of variable (V), diver­sity (D), and joining (J) regions of the gene segment through a process called V (D) J recombination. The V (D) J recom­bination is a process of selecting V, (D), and J chains in lymphocyte and arranging it into a single exon and generate a novel amino acid sequence for the antigen-binding regions (V regions) for immunoglobulins and TCR. The successful assembly is called a productive rearrangement and is moni­tored in each developmental stages and acts as a signal to progress into the next stage.

4.2.5.2.1 B Lymphocyte Development

In the bone marrow, CLP differentiated into pro-B cells without the antibody expression. The V (D) J recombination for immunoglobulins starts during this stage due to expres­sion of recombination activating gene (Rag gene). The pro-B cells differentiated into pre-B cells once the V (D) J recombi­nation for heavy chains is completed. In pre-B stage, the V (D) J recombination for light chains starts and once it is completed the pre-B cells are transformed to immature B cells. The immature B cells express IgM. The immature B cells then undergo negative selection to test its reactivity against self-antigens. The auto-reactive B cells either undergo apoptosis through a process called clonal deletion or there is reactivation of Rag for light chain receptor editing. The mature B cells thus ultimately produce and migrate to secondary lymphoid organs and express both IgM and IgD molecule. The time required to form mature B cells from hematopoietic stem is usually 1-2 weeks.

4.2.5.2.2 T Lymphocyte Development

The precursors of T lymphocyte are pro-T cells. Pro-T cells are differentiated into pre-T cells after V (D) J recombination of β chain of TCR. The pre-T cells express only β chain and further differentiate to form double positive T cells with both α and β chain along with CD4 and CD8 over their surface. Then they undergo positive and negative selection to form mature T cells.

4.2.6 Thrombopoiesis

The precursors of the platelets are megakaryocytes derived from bi-potent common myeloid progenitors (CMP) for both erythrocytes and platelets. CMP can be detected in the yolk sac of mouse embryo as early as day (E) 10.5. CMP differentiated into megakaryocyte colony forming cells (Meg-CFCs) with fetal liver by day (E) 11.5 which is the predominant source of megakaryocyte progenitor that pro­duce platelets of larger size with less cytoplasmic granules. Megakaryocytes undergo nuclear endomitosis (DNA replica­tion without cell division), disassembling of centrosomes and the translocation of microtubules to cell cortex and forms broad pseudopodia to become proplatelets in which the nucleus is extruded. A megakaryocyte can give rise to 10-20 proplatelets which protrude from megakaryocytes. The maturation of megakaryocytes to platelets required around 5 days in human and 2-3 days in rodents. Platelets may survive in blood stream for 7-10 days in human and 4-5 days in rodents.

4.3