INTRODUCTION — The circulating blood cells are formed in bone marrow through a process called hematopoiesis. The bone marrow has an enormous production capacity; it is estimated that 1010 erythrocytes and 108 to 109 leukocytes are produced per hour in the steady state. Furthermore, while cell numbers are maintained within fairly narrow limits, they can be greatly amplified on demand.
These huge cell numbers are immediate descendants of maturing precursor cells that arise from a smaller pool of progenitor cells. The progenitor cells arise from an even smaller pool of hematopoietic stem cells (HSCs), which are thought to be mostly in a resting or non-dividing state and have the capacity to self-renew (and thus maintain their numbers).
HSCs are multipotent and have the capacity to differentiate into the cells of all 10 blood lineages: erythrocytes, platelets, neutrophils, eosinophils, basophils, monocytes, T and B lymphocytes, natural killer cells, and dendritic cells (figure 1) [1-3].
This topic reviews the hematopoiesis and the regulation of HSCs. A general discussion of other types of stem cells that includes a discussion of induced pluripotent stem (iPS) cells is presented separately. (See "Overview of stem cells".)
BONE MARROW ANATOMY AND MICROENVIRONMENT
Sites of hematopoiesis — The relative red (active) marrow space of a child is much greater than that of an adult, presumably because of the high requirements for red blood cell production during neonatal life. While several sites of hematopoiesis remain active through life, including vertebrae, the sternum and manubrium, pelvic bones, and metaphyses of long bones, during postnatal life red blood cell demand and therefore production is reduced, and much of the marrow space is slowly and progressively filled with fat, in particular, the marrow in the facial bones as well as the diaphyses of long bones such as the radius, ulna, femur, and fibula [4]. In certain disease states that are usually associated with anemia (eg, primary myelofibrosis, infiltrative diseases of the bone marrow such as granulomas or metastatic cancer, or diseases characterized by ineffective erythropoiesis such as thalassemia major), hematopoiesis may return to its former sites in the liver, spleen, and lymph nodes and may also be found in the adrenal glands, cartilage, adipose tissue, thoracic paravertebral gutters, and even in the kidneys.
Bone marrow microenvironment — To develop and differentiate, hematopoietic progenitor cells require both cellular and soluble growth factor support. The cellular elements are provided by the surrounding bone, bone marrow stroma (figure 2), and the microenvironment [5-9].
The microenvironment of the bone marrow cavity is a vast network of vascular channels (sinusoids that drain into the central vein and arterioles) between which float fronds of hematopoietic cells, including fat cells. Reticular cells form the adventitial surfaces of the vascular sinuses and extend cytoplasmic processes to create a lattice on which blood cells are found. The lattice itself can be demonstrated by reticulin stains of marrow sections, while the conformation of the meshwork of reticulin and the location of hematopoietic cells in the network of vascular sinuses is illustrated by scanning electron microscopy.
The hematopoietic stem cells (HSCs) and progenitor cells are supported by a stromal cell network that provides cell-cell contact support. The network provides two major functions: an adhesive framework onto which the developing cells are bound, and the production of essential hematopoietic growth factors [10]. Along with T lymphocytes, these cells produce a variety of adhesion molecules and hematopoietic growth factors or cytokines that are thought to support the survival, proliferation, and differentiation of HSCs and progenitors. Primitive mesenchymal stromal cells (MSCs) are thought to have the capacity to differentiate into osteolineage cells, chondrocytes, adipocytes, and perivascular cells. These are referred to as CXCL12-abundant reticular (CAR) cells; they have been detected by expression of CXCL12, nestin, and leptin receptor (LEPR).
The bone marrow circulation comprises central and radial arteries that ramify in the cortical capillaries, join the marrow sinusoids, and drain into the central sinus. Cells egress from the marrow sinusoids into the venous circulation through concomitant veins. The inner, or luminal, surface of the vascular sinusoids is lined with endothelial cells, the cytoplasmic extensions of which overlap, or interdigitate, with one another. The escape of developing hematopoietic cells into the sinus for transport to the general circulation occurs through gaps that develop in this endothelial lining and through endothelial cell cytoplasmic pores.
HSCs are found in two distinct niches: an endosteal niche where HSCs lie in close proximity to a subset of spindle-shaped, N-cadherin-positive osteoblasts; and a vascular niche lined by sinusoidal endothelial cells and mesenchymal stromal cell derivatives (figure 2). While these separate anatomical regions were postulated to have different roles in supporting stem cell function, other data suggest that the role of the endosteal niche is indirect, through secretion of cytokines and extracellular matrix proteins by cells of an osteoid lineage that affect sinusoidal stromal cell function. Fluorescence staining of CD150-positive, CD48-negative, CD41-negative, lineage-negative HSC in sections of bone marrow show that most HSC localize to sinusoids in close proximity to mesenchymal stromal cells [11]. Data using alpha-catulin-GFP as a stem cell marker and deep confocal imaging showed that HSC are more common in the central marrow rather than near the endosteal surface, and that these cells are in close proximity to leptin receptor and CXCL12-positive niche cells that are close to sinusoidal blood vessels [12]. Together, the niches provide a microenvironment for cell contact and secretory functions of osteolineage cells, osteoclasts, sinusoidal endothelial cells, macrophages, MSCs including CAR cells, sympathetic neurons, and the extracellular matrix.
In addition, there are nestin-positive stromal cells, thought to be neural crest-derived, that support hematopoiesis:
●Osteolineage cells are required for G-CSF mediated HSC mobilization [13] and are important for lymphoid T and B cell development [14,15].
●Ablation of CAR cells leads to reduction of SCF and CXCL12, with decreased proliferation of lymphoid and erythroid progenitors [16,17].
●Endothelial cells express adhesion molecules such as E-selectin and secrete paracrine growth factors and cytokines: they play a role in the expansion of HSC in culture by Notch ligand production [18].
●Macrophages (CD169-positive) secrete oncostatin-M, which in turn stimulates nestin-positive cells to express CXCL12; binding of CXCL12 to the CXCR4 receptor on HSCs is important for the retention of HSCs in the bone marrow niche [19,20].
●Sympathetic nerve innervation of the bone marrow is thought to regulate the circadian cycle of HSC release from the bone marrow. Norepinephrine release is transmitted to MSC by beta3-adrenergic receptors, which leads to rapid down regulation of CXCL12 expression and HSC release [21].
HEMATOPOIETIC STEM CELLS
Properties of HSCs — Critical properties of HSCs are the capacity to self-renew and multipotency, which confers the potential to maintain long-term multilineage hematopoiesis throughout the lifespan of an individual.
The concept that sustained hematopoiesis comes from multipotent stem cells derives from the observation that mice can be protected from the lethal effects of whole-body irradiation by exteriorization and shielding of the spleen [22]. This protective effect was shown to be cell-mediated as the injection of spleen cells could initiate recovery and reestablish multilineage hematopoiesis in irradiated animals [23].
Compatible with the hypothesis of the clonal nature of hematopoiesis and the concept that a single multipotent stem cell has the capacity to repopulate the entire hematopoietic system was the demonstration that colonies of murine hematopoietic cells could be observed in the spleen of irradiated, transplanted recipients within 10 days after the transplant [24]. These spleen colony-forming units (CFU-S) generated colonies that contained precursors to erythrocytes, granulocytes and macrophages, and megakaryocytes.
Subsequent experiments using karyotypically marked donor cells confirmed the clonal origin of the differentiated cells in the colony, proving that a single multipotent HSC had given rise to these differentiated cells [25]. It was also shown that each colony that formed at day 12 after transplantation (CFU-S12) contained a number of cells that could again form a colony of differentiated progeny in a second irradiated recipient, demonstrating their self-renewal capacity.
The demonstration of a multipotent stem cell in adult bone marrow led to a systematic search for the ontogenic origins of HSC. In mammals, hematopoiesis occurs in three sequential waves, two in the yolk sac and the third intraembryonically from endothelial cells in the aorta/gonad/mesonephros (AGM) region, placenta, and other blood vessels.
Murine yolk sac hematopoiesis occurs in two waves, primitive erythropoiesis (E7.25) that comprise macrocytic nucleated erythroid cells, macrophages and megakaryocytes, followed shortly thereafter (E8.5) by definitive erythroid-myeloid progenitors (EMPs) that by E10.5 transition to the fetal liver after the circulation is established. The progeny of the EMPs support fetal erythropoiesis, during the rapid erythroid expansion during gestation, and to megakaryopoiesis and myelopoiesis (neutrophils and macrophages); the monocyte progenitors give rise to long-lived (adult) resident macrophage derivatives in brain (microglia), skin, and liver [26,27].
Stem cells from the AGM are generated from hemogenic endothelial cells and migrate to the fetal liver and finally the bone marrow [28]. The important observation that cells with long-term repopulating (LTR)-HSC activity arise first in the AGM region and not in yolk sac or other intraembryonic regions supports much evidence that HSC capable of reconstituting hematopoiesis form first within the embryo proper [29,30].
Sites of HSCs — A number of early hematopoietic cells, including the multipotent stem cells and certain committed progenitor cells, have been demonstrated in the circulation of humans and/or experimental animals [31-34].
HSCs can also be found in the umbilical cord blood obtained from the placenta directly after delivery. Cord blood is enriched in stem cells compared with bone marrow or the peripheral circulation and can be used as a source for stem cell transplantation. (See "Collection and storage of umbilical cord blood for hematopoietic cell transplantation" and "Hematopoietic cell transplantation (HCT): Sources of hematopoietic stem/progenitor cells".)
After birth, the majority of HSCs are thought to reside predominantly in the bone marrow. Gene expression profiling of primary CD34+ cells residing in the bone marrow and blood has revealed significant differences, with the marrow-derived cells cycling more rapidly, while the circulating CD34+ cells consist of a higher number of quiescent stem and progenitor cells [35,36]. These differences, although of interest, could reflect the heterogeneity of the two populations, since stem cells able to reconstitute hematopoiesis comprise only a tiny subset of each population. Practically, bone marrow is the preferred source of stem cells for transplantation of patients with bone marrow failure, while mobilized stem cells collected by apheresis are frequently used for other indications such as transplantation for hematologic malignancies.
Identification of stem and progenitor cells — Stem cells are identified by a number of assays. The factors that regulate production are not completely understood but include a variety of humoral or marrow stroma derived regulators.
Maturing precursors and mature cells are recognizable under a microscope. In contrast, progenitors and HSC are morphologically indistinguishable, and functional assays or identification of surface antigenic characteristics are required to detect their presence. Progenitor cells will form colonies of maturing cells of different lineages if they are immobilized in a semisolid medium containing methyl-cellulose or agar and stimulated with growth factors. They are often collectively referred to as colony forming cells (CFC) or units (CFU). Examples of growth factors include (table 1):
●Stem cell factor (SCF, also called Steel factor or KIT ligand) [37], interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (EPO) drive the proliferation and maturation of primitive erythroid burst-forming units (BFU-E); the more mature erythroid colony-forming units (CFU-E) are entirely EPO dependent for their first few divisions.
●SCF, granulocyte colony-stimulating factor (G-CSF), and GM-CSF drive the development of granulocyte and macrophage colony-forming units (CFU-GM).
In vitro assays — Basic and translational research using in vitro and in vivo assays discussed below have been critical in defining stem cell properties.
However, establishing these assays in clinical hematology laboratories is not justified by their expense and the infrequency with which they would be used.
Many in vitro assays that measure functions characteristic of HSC have been proposed as surrogate HSC assays but, until homogeneous populations can be evaluated in both in vitro and in vivo transplantation assays, it will be impossible to determine the precise cell type measured by these methods. Multipotency is demonstrated by colonies in semisolid media that contain granulocytes, erythrocytes, monocytes and megakaryocytes (CFU-GEMM) in methyl-cellulose cultures of human bone marrow [31,38-40].
Historical evidence for the presence of multipotent HSC was also derived from the human "Dexter" technique [41] for liquid culture of marrow in which myeloid progenitors (mostly CFU-GM) are sustained for approximately two months on and within an adherent stromal monolayer [42,43]. The progenitors can be detected by replating into methylcellulose with several growth factors at five to eight weeks, thereby demonstrating that both unipotent and multipotent cells are generated in this culture system.
This long term culture technique has been adapted to a limiting dilution assay in which long-term culture initiating cells (LTC-IC) can be quantitated after culture at different concentrations on a stromal layer for five weeks followed by replating in methyl-cellulose to score for the number of wells that do not contain colonies [44].
While these in vitro assays measure properties attributable to HSCs such as multipotency or great proliferative capacity, they cannot address the essential property of the ability of HSCs to reconstitute long-term hematopoiesis. This property ideally requires an in vivo transplantation assay. A competitive repopulation assay, based upon transplanting an unknown stem cell population together with a standard dose of competing cells into irradiated recipient mice, has been developed in the mouse to quantitate murine HSC [45]. This assay has been critical in knockout experiments to investigate the role of genes essential for normal HSC self-renewal and differentiation functions. Human HSC populations can be injected into severe combined immunodeficient (SCID or NOD/SCID) mice to demonstrate evidence of engraftment in the bone marrow [46].
It is believed that this SCID repopulating cell (SRC) represents a cell closer to the HSC than cells measured in in vitro assays [46].
Cell surface antigenic markers — Cell surface characteristics have been used to purify or define populations of cells that include HSC and progenitors. For humans, the most frequently used markers are the CD34 antigen, expressed on HSC and progenitors, and CD38, expressed on a subset of more mature progenitors and on maturing cells, but not on HSC.
The most primitive human hematopoietic cells are CD34+ and CD38- [47-49]. However, CD34+ cells comprise approximately 2 to 5 percent of nucleated cells in bone marrow, while HSC are much more rare (approximately 1 per 20,000 bone marrow cells). Thus, while markers such as CD34 are useful to enrich for primitive cells, their presence is only an indirect indication of the presence of HSC.
Transplantation of highly enriched human HSC into immunodeficient mice has been achieved with a purification that exploits a different marker profile: CD34+, CD38-, CD45RA-, CD90+ (Thy1+), and CD49f+, with further enrichment (capacity for single cell engraftment) in a Rho- fraction of these cells [50,51]. In the mouse, the state of the art for HSC purification includes the following marker profile: CD34-, CD150+, CD48-, CD41-, flt3-, and CD49b lo [52,53].
HSC heterogeneity — Hematopoietic cell culture and transplantation studies have provided a model of the stem cell compartment in which there is a continuum of cells with decreasing capacities for self-renewal, increasing likelihood for differentiation, and increasing proliferative activity. Cells progress in a unidirectional fashion in this continuum [54,55].
It is the most primitive cells with the greatest self-renewal capacity that reconstitute long-term hematopoiesis after transfer into irradiated recipient mice. These cells, termed long-term reconstituting hematopoietic stem cells (LT-HSC), can be separated from cells capable of short-term hematopoiesis (ST-HSC) or multipotent progenitors (MPP) and progressively restricted lineage committed progenitors.
However, purification of murine hematopoietic stem cells (HSC) on the basis of surface antigen expression (Thy-1.1-low,Lin-,Sca-1+) and Rh-123-low has shown that as few as five cells can confer long-term repopulation, but that heterogeneity is still present, since cells with LT-HSC activity alone or with both LT-HSC and ST-HSC potential reside in this cell fraction [56].
Transplantation studies using retrovirally marked HSC have shown long-term persistence of relatively few clones capable of multilineage differentiation. However, transplantation requires myeloablation and most likely induces stress in the transplanted HSC. Experiments using transposons to label multiple hematopoietic cells in vivo with unique genetic markers known as "barcodes" have provided major insights into steady state non-stressed hematopoiesis; longitudinal analysis shows that hematopoiesis is maintained by thousands of clones, many with properties of ST-HSC and progenitors [57]. Tracing of LT-HSC fates using Tie2Cre to label the most primitive HSC during embryonic development also suggests that ST-HSCs make a more substantial steady state contribution to hematopoiesis than LT-HSCs, and that only approximately 30 percent of the latter cells are productive during the steady state [58].
The traditional model of a hierarchy of differentiation from LT-HSC through the multipotent progenitor (MPP) to oligopotent common myeloid and common lymphoid progenitors (CMP and CLP), with CMPs branching to megakaryocyte erythroid (MEP) and granulocyte/monocyte progenitors (GMP) has been challenged. These multipotent progenitors were analyzed with additional markers that demonstrated heterogeneity within each of these populations. Single cell sorting and analysis of progeny after culture in multiple cytokines showed that during fetal development, megakaryocyte and erythroid commitment occurs at multipotent, oligopotent and unipotent stages of differentiation, while myeloid and lymphoid/monocyte progenitors arise at the latter two stages only. In contrast, adult bone marrow is dominated by unilineage progenitors of mostly myeloid and erythroid potential, while megakaryocyte commitment occurs directly at the multipotent HSC level [59].
Transcriptional studies of hematopoietic cells (single cell RNA sequencing [scRNA-seq]) have confirmed the idea that different progenitor populations are heterogeneous at the transcriptional level. This approach has identified single HSCs and MPPs that co-express gene sets specific for different unilineage programs [60,61]. Epigenetic data from single cell transposase accessible chromation (scATAC-seq) for open accessible chromatin in human fetal liver cells indicate that this lineage priming occurs at the epigenetic level [62].
The ability to analyze single stem and progenitor cells defined by cell surface markers has determined that megakaryocyte commitment occurs at the HSC level [59]. Furthermore, single cell genomic studies show that stage specific cells can coordinately express marker genes specific for distinct unilineage programs, and that this lineage priming occurs at transcriptional level (shown by scRNA-seq) and the epigenetic level (shown by scATAC-seq) [60-63]. The conventional way of depicting stages of hematopoiesis as discrete steps (figure 1) may in fact be more of a continuum, with heterogeneity at each step [64]. What determines the final lineage fate is still an open question and is thought to be a stochastic process.
STEM CELL REGULATION
Regulatory molecules — Most of the regulatory molecules are produced by hematopoietic accessory cells in close proximity to progenitor cells. These molecules are produced as part of an incompletely understood complex regulatory network operating at close range and may involve accessory cell-progenitor cell interactions. Stem cell factor (SCF) and FLT3 ligand (FLT3L), both receptor tyrosine kinase ligands, interact with a variety of hematopoietic progenitor cells, perhaps most importantly with very early stem cell populations. Both of these factors are expressed as membrane-bound and soluble forms, the former consistent with the importance of close-range interactions in the bone marrow microenvironment.
Other stem cell factors that affect HSC homeostasis include Wnt, Jagged, bone morphogenic protein (BMP), and angiopoietin-like growth factors.
●Wnt signals via canonical (beta-catenin) and noncanonical pathways. While overexpression of Wnt3A leads to expansion of the HSC pool, constitutively activated beta-catenin can lead to aplastic anemia [65,66]. These differences may be reconciled by different roles for the canonical and noncanonical pathways in HSC activation and quiescence, respectively [67].
●Jagged2 appears to be the predominant Notch ligand presented by the osteoblast; it leads to HSC expansion through Runx1 in a zebrafish system.
●BMP induces HSC production in zebrafish and Xenopus systems.
●Angiopoietins can also expand HSC populations [68].
●Other growth factors such as osteopontin and TGF beta are negative regulators and may be important in maintaining stem cell quiescence.
Self-renewal versus differentiation — The formed elements of the blood in vertebrates, including humans, continuously undergo replacement to maintain a constant number of red cells, white cells, and platelets. The number of cells of each type is maintained within a narrow range in a typical adult – approximately 5000 granulocytes, 5 x 106 red blood cells, and 150,000 to 300,000 platelets per microliter of whole blood. Regulatory mechanisms that maintain a balanced production of new blood cells are far from completely understood, but present evidence strongly supports the following basic principles (figure 1):
●A single multipotent stem cell is capable of giving rise to many committed progenitor cells, differentiated recognizable precursors of the specific types of blood cells, and mature cells by clonal proliferation. Furthermore, most multipotent stem cells are quiescent, and hematopoiesis appears to be sustained in the steady state by the progeny of only a small number of activated stem cells. The "clonal succession" hypothesis that a series of stem cells may contribute clones successively to maintain hematopoiesis throughout the life span of an individual is an attractive explanation for the existence of "active" and "resting" stem cell clones, and has strong but not unequivocal experimental support [69]. In vivo labeling data with bromodeoxyuridine (brdU) suggest that approximately 8 percent of murine stem cells enter the cell cycle every day [70], making it appear more likely that stem cells regularly make the transition from a resting to an activated state.
●The multipotent stem cell is capable of self-renewal, but this property may not be unlimited [56,71,72]. Instead, the potential of HSC to self-renew is finite, determined by replicative history [73,74] and perhaps also by accumulation of damage to DNA [75]. In addition to their limited proliferative potential, committed progenitors also "die by differentiation," and their numbers depend upon influx from the multipotent stem cell pool [76].
●Committed progenitor cells are capable of response to humoral or marrow stroma derived regulators, some produced in reaction to the circulating levels of a particular differentiated cell type (see "Regulation of erythropoiesis" and "Regulation of myelopoiesis"). In this response, the progenitor cells proliferate and differentiate to form the recognizable blood cells. Under this type of control, amplification of production occurs at the committed progenitor cell level. There is evidence that committed progenitors can self-renew. For example, BFU-E can increase in number under the influence of glucocorticoids [77], which may underlie the rapid increase in erythropoiesis during stress.
Two models have been proposed to explain the mechanisms that influence the choice of stem cells between self-renewal and differentiation (ie, lineage commitment). In one, self-renewal or differentiation is considered to occur in a random or stochastic manner, only dictated by a certain probability [76,78]. A contrasting model proposed that the hematopoietic microenvironment is inductive, based upon the observation that domains of a given lineage exist within single spleen colonies [79]. In this model, the hematopoietic inductive microenvironment (HIM) was thought to direct stem cell fate. However, the preponderance of differentiating cells of one or other type within colonies appears to result from events that affect maturation rather than lineage commitment.
There is now much evidence that supports the stochastic or random model for both lineage commitment and restriction of differentiation potential [80-82]. In this model, combinations of nuclear transcription factors have been characterized that play a role in the specification of stem cells from mesenchymal cells and in early stem cell proliferation, while other unique sets of transcription factors appear to be activated, perhaps randomly. These transcription factors are essential for the development of the different cell lineages because they dictate the synthesis of lineage specific proteins such as cell surface growth factor receptors [83-85]. The possible role of extrinsic factors (such as leukopenia or anemia) in the regulation of stem cell levels is uncertain [86,87].
The expression of transcription factors and the critical genes regulated by them during hematopoiesis are dependent on the state of the surrounding DNA and are influenced by epigenetic changes (eg, variation in DNA and chromatin that does not involve sequence change) [88]. Methylation of cytosine in the context of CpG dinucleotides is associated with transcriptional repression, while unmethylated CpG islands are found in actively transcribed promoters. A variety of post-translational modifications to histone tails correlate with distal enhancer and promoter activation and repression. Changes in DNA methylation and histone modifications are important in the regulation of HSC self-renewal and differentiation as well as the developmental changes that occur with fetal development and stem cell aging [89,90]. The DNA methyl transferase Dnmt3a is critical in silencing HSC self-renewal genes enabling differentiation [91]; histone demethylases such as Lsd1 and Jarid1b play similar roles [92,93].
Role of HSCs and the stem cell microenvironment in aging, inflammation, and myeloid malignancy — Cellular features of aging in mouse models have shown an expansion of the stem cell pool with decreased homing and repopulation capacity, anemia, and a myeloid differentiation bias.
Cell-intrinsic defects include:
●Genomic instability leading to age-related clonal hematopoiesis (ARCH) and metabolic deregulation. While normal quiescent HSCs depend on glycolysis, during activation and differentiation HSCs undergo a metabolic shift to oxidative phosphorylation and generation of reactive oxygen species. Aged HSCs show a shift from quiescence to activation, and differentiation and stem cell reconstitution decline.
●These changes are associated with epigenetic alterations that involve sirtuins, histone acetyl transferases, dioxygenases such as methylcytosine dioxygenase 2 (TET2) [94], and DNA methyltransferase 3 alpha (DNMT3A). Interestingly, Dnmt3a loss immortalizes HSCs in mice [95,96]. Cell-extrinsic aging defects include loss of endosteal osteoblasts, osteoprogenitor cells, and sympathetic nerves important for maintenance of quiescent HSCs.
●Sinusoidal endothelial cells (ECs) and mesenchymal stromal cells (MSCs) show downregulated expression of HSC supporting factors such as SCF, CXCL12, and ANGPT1 [97]. Importantly, young ECs can rejuvenate aged HSC in vitro and in vivo [98].
In response to acute inflammatory signals in the setting of infection or tissue injury, HSCs lose quiescence and transiently proliferate in response to cytokines and Toll-like receptor (TLR) ligands. Upregulation of TLR4 and myeloid differentiation primary response (MyD88) by endothelial cells, and subsequent secretion of G-CSF, results in emergency granulopoiesis. Single-cell RNA studies showed that loss of the endothelial expressed Notch ligand DLL4 results in a bias towards myelopoiesis [99]. However, chronic exposure to inflammation may be deleterious and lead to stem cell depletion and exhaustion [100]. Interferons such as IFN-gamma can affect HSC self-renewal and repopulating function by modulating their response to cytokines such as STAT1 [101].
Cells that comprise the HSC niche may also induce irreversible changes in HSCs. The first example of this surprising mechanism came from study of retinoblastoma (RB) protein or retinoic acid receptor gamma (RARγ) deficient mice. Surprisingly, transplantation of wild type HSCs into these mice led to the development of myelodysplasia in the donor cells [102,103]. Subsequent studies found that deletion of the microRNA processing enzyme Dicer in osteoprogenitor cells led to myelodysplasia and AML. The Dicer deletion reduced the expression of the SBDS gene (the gene mutated in the inherited bone marrow failure disease Shwachman Diamond syndrome [SDS]) [104]. Patients with SDS have an increased risk of developing myelodysplasia and AML. Additional studies demonstrated that an activating mutation of beta-catenin in MSCs resulted in the development of MDS and AML through Jagged 1 expression and Notch signaling in HSCs [105]. Furthermore, mutations in PTPN11 in mesenchymal precursor cells in the context of Noonan syndrome promoted a juvenile myelomonocytic leukemia (JMML)-like disease and caused donor-derived myeloproliferative neoplasia in donor-derived cells after transplantation [106].
Several review articles address these interesting roles of the bone marrow microenvironment [107-111].
THERAPEUTIC USES OF HSCs — A cell with the characteristics of a self-renewing multipotent stem cell has not been clearly defined in humans. However, the presence of stem cells capable of long-term hematopoietic reconstitution (LT-HSC) is inferred from the success of transplantation, using bone marrow, chemotherapy and/or G-CSF mobilized blood, or umbilical cord blood as a source of stem cells in humans. (See "Donor selection for hematopoietic cell transplantation" and "Hematopoietic cell transplantation (HCT): Sources of hematopoietic stem/progenitor cells".)
The major therapeutic uses for stem cells are:
●Transplantation of allogeneic stem cells to reconstitute hematopoiesis in patients with either bone marrow failure or genetic disease affecting blood cell production or function. (See "Treatment of aplastic anemia in adults" and "Hematopoietic cell transplantation for aplastic anemia in adults" and "Hematopoietic cell transplantation for acute myeloid leukemia and myelodysplastic syndromes in children and adolescents", section on 'Introduction' and "Treatment of acquired aplastic anemia in children and adolescents", section on 'Introduction'.)
●Transplantation of autologous or allogeneic stem cells to allow reconstitution of hematopoiesis in patients who might benefit from intensive radiochemotherapy for the treatment of malignant disease (eg, acute myeloid leukemia). (See "Acute myeloid leukemia in younger adults: Post-remission therapy" and "Acute myeloid leukemia: Management of medically unfit adults".)
●Insertion of normal gene copies or transcriptional repressors into genetically defective stem cells, which could then be transplanted and expressed long-term at sufficient levels to correct or alleviate genetic disease (ie, gene therapy). Major advances are being made in this area, as discussed in detail separately. (See "Overview of gene therapy, gene editing, and gene silencing", section on 'Inherited single gene disorders' and "Overview of gene therapy, gene editing, and gene silencing", section on 'Clinical applications of gene silencing'.)
Much research focuses on identification of the optimal donor and source (bone marrow or blood) of stem cells; stem cell purification to deplete contaminating tumor cells, reduce graft-versus host disease, and also optimize gene transfer; as well as ex vivo stem cell expansion to reduce malignant cell contamination and/or the amount of marrow required for successful engraftment.
Under most circumstances the growth of LT-HSC in the marrow appears to require a microenvironmental "niche" [1,112-114]. Thus, isogeneic marrow infusions are not successful unless the recipient is irradiated or treated with sufficient doses of cytotoxic drugs to create an adequate number of "niches" (see 'Bone marrow microenvironment' above). Experimentally, however, marrow ablation is unnecessary if very large numbers of stem cells are transplanted [115].
Clinical relevance of HSC cell surface phenotype — CD34+ cell counts are used clinically to determine whether sufficient HSCs for engraftment have been obtained after mobilization or collection [116]. These mobilized HSCs have been used for:
●Allogeneic transplants in leukemia
●Autologous transplants in lymphoma or multiple myeloma
●Umbilical cord blood transplants
CD33 is a transmembrane glycoprotein expressed on maturing myeloid cells but not HSCs. Its expression at high levels in leukemic myeloblasts has led to methods to target these cells with antibodies linked to cytotoxic conjugates. Gemtuzumab ozogamycin was the first antibody drug conjugate (ADC) to be studied. Other conjugates have been developed, as have CD33 chimeric antigen receptor T cell (CAR-T cell)-based therapies.
CD117 (KIT) antibody drug conjugates are being explored for their role in HSC transplantation. Depletion of HSC by immunologic conditioning may spare mature hematopoietic cells and cause substantially less inflammation and nonspecific collateral damage to other organs compared with radiation and chemotherapy [117,118].
SUMMARY
●HSC properties – Hematopoiesis is an enormously productive and regulated process, with >10 billion erythroid and myeloid cells produced per hour in an adult, and with numbers maintained within narrow limits. This is sustained throughout the lifespan of an individual by a slowly cycling quiescent pool of long-term hematopoietic stem cells (LT-HSC) that differentiate into multipotent progenitor cells or short term HSC (ST-HSC). Stem cells have the capacity to self-renew and to commit to one of the 10 hematopoietic lineages by a stochastic process that induces a proliferation and differentiation program driven by expression of sets of transcription factors. (See 'Properties of HSCs' above.)
●Bone marrow anatomy – Adult HSC in bone marrow reside in endosteal and vascular niches on a lattice of reticular cells. The niches provide a microenvironment for cell contact and secretory functions of osteolineage cells, osteoclasts, sinusoidal endothelial cells, macrophages, mesenchymal stromal cells, sympathetic neurons, and the extracellular matrix. (See 'Bone marrow anatomy and microenvironment' above.)
●Role of stromal cells and growth factors in differentiation – Major advances in cell sorting and genomics have enabled the interrogation of HSC and progenitor cells at the single cell level for differentiation outcomes, gene expression, and regulation. These studies have led to the idea that changes in stages of differentiation are more of a gradual continuum than one of discrete definitive steps, with heterogeneity of single cells at the stem cell and multipotent progenitor cell levels simultaneously primed to commit to different lineages. What regulates the commitment to a particular lineage is still an open question.
It appears likely that two types of extrinsic control can affect stem cell regulation. At one level is control by stromal cells, perhaps primarily on stem cell survival but also on their response to humoral factors. At a second level are the humoral growth factors, which appear to affect stem cells but have a greater effect on the amplification and differentiation of the committed maturing progenitor cells and precursor cells. (See 'Stem cell regulation' above.)
●Clinical implications – Application of knowledge regarding stem cell properties facilitates their identification from bone marrow, peripheral blood mobilization, or umbilical cord blood. HSC and progenitor cell characterization has also led to clinical trials to devise optimal methods to target CD33, expressed on leukemic blasts, and to exploit CD117 (KIT), the stem cell factor receptor on HSC, to deplete these cell as an alternative to conventional radiation and/or chemotherapy before HSC transplantation. (See 'Therapeutic uses of HSCs' above.)
20 : Bone Marrow Macrophages Contribute to Diabetic Stem Cell Mobilopathy by Producing Oncostatin M.
97 : Adrenergic nerve degeneration in bone marrow drives aging of the hematopoietic stem cell niche.
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