The hematopoietic system, which comprises all the cellular components of the blood, is one of the earliest organ systems to evolve during embryo development. Hematopoietic stem cells (HSCs), which are rare blood cells residing in the bone marrow of the adult organism, are the founder cells that give rise to the entire hematopoietic system. HSCs are primarily characterized by their ability to self-renew, as well as their potential to mature and differentiate into all blood cell lineages, including erythroid, myeloid, and lymphoid cells. Considering the short lifespan of mature blood cells (around 120 days in humans), the self-renewing and multi-potent nature of HSCs provides a way to continuously replenish the mother blood cell population that can give rise to more differentiated lineages throughout life .
During early development, the various cell types of the hematopoietic system are formed at distinct anatomical niches within the embryo, in a spatially and temporally controlled manner, until this function is completely taken over by the bone marrow and thymus (for T-lymphoid cell generation) just prior to birth. A number of studies have now confirmed that the development of the hematopoietic system, in humans and other mammals, occurs in two phases: a primitive hematopoietic phase that gives rise to transitory, bi-potent HSCs, and a definitive hematopoietic phase that generates long-lived, multipotent HSCs .
Primitive hematopoiesis: The primitive phase of hematopoiesis starts very early, at around the third week of mammalian embryo development, in an extraembryonic tissue called the yolk sac. Within this yolk sac, mesodermal cells start forming cell aggregates at around day 16 of embryo development . Soon after, the peripheral cells of the aggregate acquire endothelial characteristics, while the inner cells disappear to form the lumen of the primitive blood vessels. At around day 19, distinct blood islands are formed by mesodermal cells that remain attached to the endothelial walls, and cells of these blood islands give rise to progenitors of the erythroid and myeloid lineage . Upon the separation of yolk sac from the embryo at the 19-day stage, when the blood circulation between the extra- and intra-embryonic compartments is still not established, only erythroid and myeloid cells were identified in the yolk sac. This study confirmed that the primitive hematopoietic cells generated in the yolk sac lack the potential to differentiate into a lymphoid lineage . The simultaneous emergence of both endothelial and hematopoietic cells, as well as the expression of common molecular markers and transcription factors  by both of these cell types confirmed that they arose from a common mesodermal ancestor called the hemangioblast, whose existence was initially proposed in the first half of the twentieth century [Murray PDF, 1932].
Definitive hematopoiesis: The earliest evidence for intraembryonic hematopoietic activity came from experiments in birds, in which blood cell progenitors were identified in and around the region neighboring the dorsal aorta . In human and other vertebrate embryos, a homologous region called the aorta-gonad-mesonephros (AGM) as well as the earlier stage precursor tissue, the Para-aortic Splanchnopleura (P-Sp), were identified as the primary sites for definitive hematopoiesis during early embryogenesis. HSCs could be detected in the P-Sp and AGM regions as early as day 19 of gestation; these HSCs showed the potential to form progenitors of both myeloid and lymphoid lineages, unlike the yolk sac HSCs, whose differentiation potential was restricted to erythroid and myeloid lineages . The central role of the AGM region in establishing autonomous hematopoietic activity within the embryo was further analyzed through immunohistochemical analyses of human embryos, which revealed a high concentration of CD34 (a molecular marker that identifies undifferentiated, immature progenitor cells) expressing cells in the AGM region as well as in the ventral endothelium of the aorta . The results were followed up with in vitro colony assays using a subset of CD34+ expressing cells, which demonstrated that these AGM region cells could generate high clone numbers of hematopoietic progenitors . More recent studies have employed three-dimensional culture techniques, which enable the development of organ rudiments isolated from whole embryos, to further confirm the hematopoietic potential within the human embryo .
Following their emergence from the AGM region, the definitive HSCs enter the primitive blood circulation and migrate to the other major embryonic hematopoietic sites, including the fetal liver, thymus, and spleen. The HSCs form colonies in the fetal liver by rapidly proliferating and expanding their populations, and thereafter, differentiate into erythroid and myeloid progenitors. The spleen and the thymus, both of which develop later during gestation, also function as sites for differentiation into highly differentiated lineages. During the later stages of gestation, the HSCs eventually move on to colonize the bone marrow, which then functions as the major hematopoietic site for the entirety of the adult life .
The hemogenic endothelium theory: This theory is more pertinent to the definitive hematopoiesis phase and proposes that the HSCs directly originate from a specialized vascular endothelium located in the AGM region, called the hemogenic endothelium. The earliest evidence for the existence of a specialized endothelium came from fate mapping studies using chick and mice embryos, in which endothelial cells were labeled with a marker before the onset of hematopoiesis. These markers were subsequently expressed in the hematopoietic precursors that emerged later on, suggesting that the endothelium is the ancestor for the origination of the blood-forming tissue . Concurrent with these findings, culture assays using embryonic endothelial cells confirmed the ability of these cells to differentiate into both lymphoid and myeloid progenitor cells . Similar studies were carried out using human embryonic CD34+ endothelial cells harvested from the AGM. These cells were cultured on a stromal layer to induce multi-lineage hematopoiesis, and they confirmed the existence of a hemogenic endothelium since the frequency of the endothelial cells in the AGM correlated with the extent of hematopoiesis in vitro .
The mesodermal prehematopoietic precursor theory: This theory has emerged as a combination of the hemangioblast and the hemogenic endothelial theories of hematopoiesis, and posits that both these events can happen at the same time as sequential steps of a common ontogenic pathway. According to this theory, which is based on findings from mouse embryonic stem cell differentiation studies, the hemangioblast gives rise to the hemogenic endothelium, from which the hematopoietic cells are then derived . This concept has been supported by genetic studies, which showed Runx1 (a transcription factor that promotes hematopoiesis) expression in both the aortic endothelium and the ventral sub aortic mesodermal cells  as well as by fate mapping experiments in mice embryos that confirmed the emergence of HSCs from the hemogenic endothelium lining the ventral wall of the aorta, which was in turn derived from an early mesodermal cell cluster .
Endothelial-hematopoietic transition: The emergence of the HSCs from the hemogenic endothelium is reliant on an important cellular event called endothelial-hematopoietic transition (EHT). Endothelial cells undergo a series of phenotypic and genetic alterations as they shed off from the aortic wall into the sub aortic space. During this process, endothelial cells from the aortic floor repeatedly contract and bend towards the sub aortic space, and as they do so, they gradually lose contact with their neighboring endothelial cells, round up, and attain oscillatory motion along the aortic vessel’s axis. Eventually, the cells completely delaminate from the aortic endothelial walls and move freely into the sub aortic space, at which stage they have morphological features resembling hematopoietic progenitor cells . These events were first visualized using live imaging techniques in Zebrafish embryos, and similar cellular transitions have also been observed by live imaging of cultured organotypic slices of the mouse AGM region .
Once blood circulation is established during the earliest stages of hematopoiesis, the pulsatile nature of blood flow within the aorta generates a range of biomechanical forces, such as fluid shear stress, hydrodynamic pressure, and circumferential stress. A number of studies have shown that the hemodynamic environment within the blood vessels has a direct influence on the structural and functional characteristics of the endothelial cells lining the inner walls of the vascular tissue (reviewed ).
Based on these findings, a number of research groups speculated that biomechanical forces might also be required during the development of the hematopoietc system. This was supported by the close association between the development and the anatomical locations of endothelial and hematopoietic lineage forming tissue in the embryo. When embryoid body cells derived from mouse embryonic stem cells were exposed to fluid shear stress at a magnitude that is comparable to the forces acting under physiological conditions, the embryoid body cells upregulated expression of CD31 protein, which is a molecular marker of endothelial and hematopoietic cell lineages, as well as upregulation of Runx1 transcription factor for hematopoiesis. These molecular changes had direct functional implications, since shear stress-exposed cells showed a greater tendency to form hematopoietic precursor colonies. Similar findings were obtained using in vitro cultures of the P-Sp and AGM regions, as exposure of these embryonic tissues to shear stress increased their hematopoietic potential. Moreover, these cells showed an inclination to differentiate into specific lineages, particularly B-lymphoid and erythroid lineages, under the influence of physical stimuli .
In the bone marrow, which are the long-term hematopoietic sites in adult organisms, discrete microenvironments called niches exist. The niches are defined by the mechanical properties of the matrix, and by their distinct biomolecular and cellular profiles . The dynamic interactions that occur between the HSCs and the marrow niches are responsible for maintaining hematopoietic stem cell populations as well as for fate determination of these HSCs . For instance, endosteal niches located near the bone surface contain inactive, long-lived HSCs, while perivascular niches located deep inside the marrow house active, self-renewing, short-lived HSCs . The influence of the mechanical properties on bone marrow hematopoiesis were demonstrated by a study that used extracellular matrix protein-coated polyacrylamide substrates to mimic distinct marrow niches. Mouse HSCs grown on substrates of increasing stiffness underwent a change in morphology from a circular to a polarized shape, and their rate of proliferation also increased. More interestingly, the study reported that the matrix stiffness (which was modulated by the ligand composition) had a direct impact on the lineage determination of these HSCs. Substrate stiffness was modulated depending on ligand composition. At endosteal niches, a stiffer microenvironment due to higher concentrations of fibronectin led to less-differentiated early stage hematopoietic progenitors. However, softer perivascular niches containing high levels of laminin promoted the differentiation of the progenitor cells into myeloid lineages. Intracellular tension generated by actin–myosin contractility was shown to further compliment the fate-determining capacity of the extracellular biophysical cues .
A large number of blood disorders, ranging from non-malignant conditions such as anemia and thalassemia to malignant disorders such as lymphoma and leukemia, affect the human population. It is therefore becoming increasingly essential to gain deeper insights into the mechanisms that regulate the proliferation, self-renewal, and lineage commitment of HSCs, so as to be able to better understand the pathophysiology and the molecular-level dysregulation underlying many of these conditions. The ability of HSCs to repopulate the entire hematopoietic system of an organism has made them invaluable tools for the treatment of a number of hematological disorders. In recent decades, the regeneration of the hematopoietic system through bone marrow transplantations has become a routine therapeutic approach in treating patients suffering from debilitating blood disorders . Recent research highlighting the regulatory role of the bone marrow niches during hematopoiesis have provided a basis for the in vitro development of optimized biomaterial platforms for the maintenance and differentiation of HSCs that could be used as analytical tools for monitoring real-time functional changes in HSCs.