It has been estimated that the concentration of cells within the marrow is 109/mL; as a result, multiple cell–cell and cell–matrix interactions occur. A major advance in experimental hematology has been the capacity to grow hematopoietic cells in long-term culture. When high concentrations of marrow cells are placed in serum-containing cultures, a stromal cell layer and extracellular proteinaceous matrix form, and when subsequently recharged with fresh marrow cells, these long-term cultures (LTCs) are capable of supporting hematopoiesis for months with simple demidepletion and replacement of culture medium. It is assumed that the cell–cell and cell–matrix interactions that develop in such cultures more closely resemble those found in vivo, helping to explain the longevity of such cultures and their capacity to maintain hematopoietic stem and primitive progenitor cells far longer ex vivo than do nonstromal cell-containing cultures. The molecular basis for the improved hematopoietic environment of LTCs is thought to rely on stromal cell surface molecules that promote cell–cell contact, prevent programmed cell death, and regulate growth.
The microenvironmental effects on HSCs have far reaching clinical implications as well; our ability to mobilize marrow stem cells for transplantation has greatly changed the way we treat hematologic and other malignancies, and ultimate success in the efforts of experimental hematologists to expand HSCs ex vivo with cocktails of cytokines and stromal cells for applications in gene therapy and regenerative medicine will undoubtedly derive only from a thorough understanding of the molecular bases for the interaction of HSCs with their microenvironment (Fig. 18–2).
The figure depicts multiple elements of the hematopoietic microenvironment. The niche has two major regions in the marrow, supported by osteoblasts or vascular cells. Several cell types provide cytokines that maintain osteoblasts (Ob), which, in turn, support hematopoietic stem cells (HSC) by secreting CXCL12 and other cytokines. Osteoclasts (Oc) are also shown but are of lesser importance in HSC maintenance, and may inhibit HSC survival/proliferation. Macrophages that express α smooth muscle actin (αSM) support perivascular cells, including the CXCL12 abundant reticular (CAR) cells, that, in turn, provide CXCL12 and SCF (here termed c-kit ligand [KitL]) to HSCs. In addition to paracrine support, direct perivascular cell–stem cell contact, through integrins, also support HSCs. (Reproduced with permission from Calvi LM, Link DC: Cellular complexity of the bone marrow hematopoietic stem cell niche. Calcif Tissue Int 2014 Jan;94(1):112-24.)
Marrow stromal cells influence hematopoiesis in a number of ways, by producing several cytokines that positively or negatively affect hematopoietic cell growth,139,140,141,142 including some, like SCF, that are expressed on their cell surfaces, resulting in enhanced biologic activity.143 Stromal cells are the origin of a number of extracellular matrix proteins that either directly affect hematopoietic cells, or do so indirectly by binding growth factors and presenting them in a functional context.144 They also bear the Jagged/Delta family ligands that stimulate Notch proteins to undergo cleavage and translocation into the nucleus, events that are critical mediators of cell fate decision making,145,146 including for hematopoietic cells.147 Cell–cell interactions mediated by integrins present on hematopoietic cells and counterreceptors on stromal cells are also very important for hematopoiesis.65,72 In addition to bringing hematopoietic cells into close proximity to cells producing soluble or cell-bound cytokines, and hence raising the local concentration of these growth promoting proteins, integrin engagement leads to intracellular signaling, usually promoting entry into the cell cycle and preventing programmed cell death.148 Reflecting the vital and sometimes lineage specific roles of the hematopoietic microenvironment, the extracellular matrix and stromal cells reside in a highly organized structure (Chap. 5).
Hematopoiesis is highly compartmentalized within areas of red marrow, with erythropoiesis occurring in clusters surrounding a central macrophage,149 granulocyte development associated with stromal cells,150 and megakaryopoiesis occurring adjacent to the endothelial sinusoidal cells.151 In the adult marrow, the specialized niche in which HSCs develop into differentiated progeny has been termed the hematon by Peault, a structure that includes Str01+ mesenchymal cells, desmin-positive perivascular lipocytes, Flk1+ endothelial cells, macrophages, and hematopoietic progenitors.152 From these structures can be derived all lineages of committed colony-forming cells (e.g., CFU-GM and BFU-E) and primitive cells that score positive in CAFC assays, LTC-IC, and high proliferative potential colony-forming cell assays (Chap. 5).
One consequence (or perhaps cause) of this anatomical arrangement is that the stem cell microenvironment is quite hypoxia. It is estimated that the O2 level of the stem cell niche is approximately 5 percent. The HSC response to hypoxia is discussed in “Metabolic Characteristics” above.
The marrow microenvironment is composed of multiple cell types. Fibroblasts are perhaps the best-studied of the marrow stromal cells, and can bind to primitive hematopoietic cells by engaging cell-surface integrins.153 Marrow endothelial cells also support primitive hematopoietic cells, including LTC-IC.154 The CXCL12–abundant reticular (CAR) cells, which surround the sinusoidal endothelial cells in vivo, are also likely to play the critical niche function of the vascular wall.155,156 However, based on their ability to increase the number of HSCs when experimentally increased, osteoblasts, which line trabecular bone and reside adjacent to primitive hematopoietic cells,157 are thought to provide a critical role in serving as the HSC supportive niche.158 The origin of all of these cell types is thought to reside in the mesenchymal stromal cell (MSC), a functionally defined entity that under specific conditions can be induced to form fibroblasts, endothelial cells, CAR cells, and osteoblasts, amongst others,159 and hold promise to therapeutically manipulate hematopoiesis.160 MSCs are discussed more extensively in Chap. 30.
Marrow stromal cells affect HSCs in multiple ways. Each of these cells is known to produce a number of cytokines critical for primitive and mature hematopoietic cell development. For example, although a number of organs produce TPO constitutively,161 marrow stromal cells are induced to produce the hormone in states of thrombocytopenia.162,163 Stromal cells produce SCF constitutively in both soluble and membrane bound forms,76 and FLT-3 ligand (FL) is produced both constitutively by stromal cells and lymphocytes and can be induced to high levels in the presence of pancytopenia.164
Besides growth factor production, stromal cells are also known to display counterreceptors for the integrins present on hematopoietic cells, including VCAM-1,165 interactions that promote cell survival and proliferation in several ways.166 Osteoblast-derived annexin II serves as an adhesion molecule for HSCs.167 Stromal cells also elaborate extracellular matrix components, including collagen, laminin, FN, heparins, hyaluronan, and tenascin, which display important effects on HSCs (see “Matrix Proteins” later). These substances, in turn, engage a number of HSC integrins and other cell-surface molecules, and form a solid matrix on which hematopoietic cells firmly attach. Of considerable clinical interest, it appears that interference with cell–matrix interactions,168 or digestion of the extracellular matrix itself,169 is involved in mobilizing HSCs by some agents such as granulocyte colony-stimulating factor (G-CSF) and IL-8.
It has long been known that the marrow is innervated by the autonomic nervous system,170 which influences HSCs in several ways, such as directing HSC trafficking by acting on nestin-positive microenvironmental cells.171 One or more of these functions appear to be critical for HSC homeostasis, as marrow nerve injury impairs hematologic recovery following chemotherapy-induced injury.172
The regulation of stem cell survival, proliferation, and differentiation has been difficult to address because of the rarity of stem cells and the requirement that they be assessed using cumbersome transplantation assays. Several cytokines are able to exert effects on HSCs. The pursuit of the cytokines that affect HSCs is of more than pure physiologic interest, as the availability of the right combination of such proteins could allow expansion of the cells for therapeutic use without sacrificing their pluripotent and self-renewal capacities. Three proteins—SCF, FL, and TPO—and their corresponding receptors (c-Kit, Flt3, and c-Mpl, respectively) exert important effects on the number and/or growth of HSCs both in vitro and in vivo (Table 18–1).
Table 18–1.Cytokines and Hormones Active on Stem Cells and Progenitors ||Download (.pdf) Table 18–1. Cytokines and Hormones Active on Stem Cells and Progenitors
|Cytokine ||Principal Activities |
|IL-1 ||Induces production of other cytokines from many cells, works in synergy with other cytokines on primitive hematopoietic cells |
|IL-2 ||T-cell growth factor |
|IL-3 ||Stimulates the growth of multiple myeloid cell types, involved in delayed type hypersensitivity |
|IL-4 ||Stimulates B cell growth and modulates the immune response by affecting immunoglobulin class switching |
|IL-5* ||Eosinophil growth factor and affects mature cell function |
|IL-6 ||Stimulates B lymphocyte growth; works in synergy with other cytokines on megakaryocytic progenitors |
|IL-7* ||Principal regulator of early lymphocyte growth |
|IL-9 ||Produced by Th2 lymphocytes; costimulates the growth of multiple myeloid cell types |
|IL-11 ||Shares activities with IL-11; also affects the gut mucosa |
|IL-15* ||Modulates T lymphocyte activity and stimulates natural killer cell proliferation |
|IL-21 ||Affects growth and maturation of B, T, and natural killer cells |
|SCF* ||Affects primitive hematopoietic cells of all lineages and the growth of basophils and mast. Also termed c-Kit Ligand |
|EPO* ||Stimulates the proliferation of erythroid progenitors |
|M-CSF* ||Promotes the proliferation of monocytic progenitors |
|G-CSF* ||Stimulates growth of neutrophilic progenitors, acts in synergy with IL-3 on primitive myeloid cells and activates mature neutrophils |
|GM-CSF ||Affects granulocyte and macrophage progenitors and activates macrophages |
|TPO* ||Affects hematopoietic stem cells and megakaryocytic progenitors |
|CXCL12 ||Chemokine that attracts HSCs by binding to the CXC4 receptor |
The molecule termed SCF, steel factor, mast cell growth factor, or c-Kit ligand was cloned by several groups based on its binding to a cell surface receptor encoded by the protooncogene c-Kit,76 previously identified as responsible for the severe defects in hematopoiesis, pigmentation, and gametogenesis in W mice. As the phenotype of mice bearing alleles of W was quite similar to those of steel (Sl), but in transplantation studies one strain displayed a stem cell autonomous defect (W) while the other was not (Sl), it had been hypothesized that the two genes represented the receptor for a growth factor and the cytokine itself, respectively, a tenet proven true with the cloning of SCF.
The extracellular domain of c-Kit is composed of five immunoglobulin-like domains, which leads through a typical transmembrane domain to the intracellular domain that bears a split domain-type tyrosine kinase. A single molecule of SCF binds to the first three immunoglobulin domains (D1D2D3) of two c-Kit receptors. The two D4 domains of a dimeric c-Kit receptor display substantial electrostatic repulsion toward each other, precluding the juxtaposing of the two transmembrane domains, and by extension, the two intracellular kinase domains. Once SCF binds to c-Kit the affinity for dimer formation overcomes the D4 electrostatic repulsion and the two kinase domains are brought together to initiate signaling.173 The intracellular mediators activated by SCF binding to c-Kit include phosphoinositol 3′-kinase (PI3K), mitogen-activated protein kinases (MAPKs), phospholipase C gamma (PLCγ), and c-Src (Chap. 17; reviewed in Ref. 174).
SCF is synthesized by marrow fibroblasts and other cell types. Soluble SCF is a highly glycosylated 36-kDa protein released from its initial site on the cell membrane by proteolytic processing. An alternatively spliced form of SCF messenger RNA (mRNA), that does not encode the cleavage site, remains on the cell membrane, and is a more potent stimulus of c-Kit-receptor-bearing cells.140 The ratio of soluble to membrane encoding SCF mRNA varies widely in different tissues, ranging from 10:1 in the brain, to 4:1 in the marrow, to 0.4:1 in the testis.
The importance of SCF to hematopoiesis is easily demonstrated; although nullizygous mice (Sl/Sl) are embryonic lethal because of a number of developmental defects, the presence of a partially functional allele (Sld) allows compound heterozygotes (Sl/Sld) to survive into adulthood, albeit with severe anemia because of diminished numbers/quality of HSCs. In addition to its critical role in the development of embryonic and fetal hematopoiesis, treatment of adult mice with an antibody that neutralizes the SCF receptor, c-Kit, also results in severe pancytopenia, indicating an important hematopoietic role for the receptor/ligand pair throughout life.
When present in culture SCF alone can maintain the long-term repopulating ability of murine Sca-1+/Rhlo/Lin− hematopoietic cells, suggesting that the cytokine can promote the survival of HSCs in vitro.175 However, alone, SCF is only a weak stimulator of cell proliferation, primarily inducing the development of mast cells both in vitro and in vivo. Nevertheless, in the additional presence of IL-3, IL-6, IL-11, G-CSF, or TPO, SCF exerts profound effects on the generation of hematopoietic progenitor cells of all lineages,176,177,178 pointing to primitive hematopoietic cells as critical targets. The molecular mechanisms of such synergy are beginning to emerge.179 A physical association of c-Kit and EPOR has been detected following SCF stimulation of cells bearing both receptors, an event that is essential for their functional synergy.180
FL was cloned as the binding partner for the then newly identified novel orphan receptor Flt3,181 a protein most closely related to the receptors for macrophage colony-stimulating factor (M-CSF) (hence the term flt = fms-like tyrosine kinase), and c-Kit. FL is expressed by T lymphocytes and marrow stromal cells.164,181 The Flt3 receptor is a 160-kDa cell-surface molecule expressed primarily on primitive hematopoietic cells.182 Like c-Kit, activation of Flt-3 results in activation of several signaling mediators, including the p85 subunit of phosphatidylinositol 3-kinase, SHP, PLCγ, and a guanosine 5′-triphosphatase (GTPase)-activating protein, activating Ras.183 Normal Flt3 signaling also activates the MAPKs extracellular regulated kinase (ERK)-1 and ERK2 but leads to only weak phosphorylation of signal transducer and activator of transcription (STAT)-5, in contrast to an oncogenic form of the receptor, identified in 25 percent of patients with myelodysplastic syndromes or acute myelogenous leukemia.184,185 In the leukemic cells of such patients Flt3 bears an internal tandem duplication of the kinase domain, resulting in the constitutive activation of the receptor. Clinically, this is associated with reduced likelihood of patient survival; hence, this observation has led to an attempt to control the growth of such mutant-receptor-bearing cells with specific Flt3 kinase inhibitors,186 with some success (Chap. 88).
FL was initially cloned using a soluble form of the receptor to identify ligand-bearing cells.187 As their receptors bear a number of common structural features, it was not surprising to find that FL shares significant structural homology, as well as biologic properties with both M-CSF and SCF. Like the other two cytokines, FL displays a 4α-helix bundle tertiary structure and exists in both membrane-bound and soluble states, the result of alternate splicing of the primary transcript that does or does not include a cleavage site for its release from the cell membrane.188
Unlike SCF levels that remain relatively static regardless of blood cell counts,76 blood concentrations of FL can rise more than 25-fold in response to pancytopenia.189 Interestingly, only pancytopenia, and not individual lineage deficiencies cause an increase in blood FL concentrations, suggesting that the cytokine is a bona fide regulator of stem or primitive hematopoietic cells. Consistent with this conclusion, transplantation data indicate that HSCs from Flt3-deficient mice do not effectively reconstitute the hematopoietic system,190 being three- to eightfold less efficient in repopulation as wild-type cells, a conclusion reinforced by its genetic combination with c-Kit mutant mice.190
Like SCF, FL appears to act on HSCs only in synergy with other hematopoietic cytokines,191,192 a finding particularly true for its combination with TPO.193,194 In addition, FL is a potent stimulus of B lymphopoiesis and granulocyte-macrophage proliferation and development, particularly of the latter toward the dendritic cell lineage.195,196
TPO is a 45- to 70-kDa hormone that was cloned by both traditional biochemical purification and expression cloning strategies based on the use of a then orphan class I cytokine receptor, first identified as the cellular homologue of the murine-transforming oncogene v-mp1.197 TPO bears extensive sequence homology to erythropoietin (EPO), sharing 20 percent identity and an additional 25 percent similarity. The hormone is produced in several organs, including the liver, kidney, skeletal muscle, and the marrow stroma. Based on murine liver transplantation studies about half of steady-state TPO production occurs in that organ,198 but in states of thrombocytopenia the marrow stroma increases production substantially.160,163 The hormone acts on megakaryocyte (MK) progenitors to enhance their survival and proliferation and on immature MKs to promote their differentiation, but surprisingly not on mature cells during platelet formation.199 Multiple lines of evidence also indicate that TPO can exert profound effects on the HSC. The hormone also supports the survival of candidate HSC populations, and acts in synergy with IL-3 and SCF to induce these cells into the cell cycle and increase their output of both primitive and committed hematopoietic progenitor cells of all lineages.200,201 These properties are also seen in vivo. For example, administration of the hormone to myelosuppressed animals leads to more rapid recovery of all hematopoietic lineages, including primitive cells,202,203,204,205 and genetic elimination of TPO or its receptor severely reduces the number of marrow stem and progenitor cells of all lineages to 15 to 25 percent of normal values.87,206,207 In addition, as noted in “Flt3 Ligand” above, TPO acts in synergy with FL to expand primitive hematopoietic cells in suspension culture, and when used to supplement LTC, the hormone maintained HSC numbers for up to 2 months,207 compared to standard LTCs in which repopulating HSCs are no longer detectable at this time.
The TPO receptor, the product of the cellular protooncogene c-Mpl, is a member of the cytokine receptor family that includes EPO, G-CSF, growth hormone, leptin, and many others. Upon binding TPO, the homodimeric c-Mpl activates its tethered Jak2 kinases, leading to phosphorylation of three of the cytoplasmic domain tyrosine residues. These phosphotyrosine residues then act as docking sites for several secondary signaling molecules, including STATs, MAPKs, and PI3K, ultimately leading to the expression of a number of transcription factors (e.g., homeobox-containing proteins, HIFs) and cell-survival molecules (e.g., BclXL). A more complete discussion of the molecular mechanisms by which TPO affects the HSC is found in Chap. 17.
CXCL12 (Previously Termed Stromal Cell-Derived Factor 1)
CXCL12 is produced by a number of the cells that occupy the hematopoietic microenvironment, and has profound effects on HSC localization to the stem cell niche.22 However, this chemokine is also thought to display direct effects on the survival and proliferation of hematopoietic stem and progenitor cells, both alone and in synergy with other hematopoietic cytokines.208,209
The human homologue of Drosophila Notch was identified as an altered gene product in T-cell leukemia.210 The discovery that the hematopoietic microenvironment displays Notch ligands, and that Notch isoforms appear on primitive hematopoietic cells,211,212 opened the possibility that Notch affects HSCs. This assertion has been directly proven: The Notch ligands Delta1 and Delta4 expand primitive hematopoietic cells.213,214 It is possible that the favorable effect of marrow osteoblasts on HSCs is a result of their expression of Notch ligands, as inhibition of Notch processing blocks the expansion in HSCs seen in mice in which osteoblasts have been experimentally expanded.158
A role for Wnt proteins in hematopoiesis was suggested by their localization at sites of fetal blood cell production and their ability to expand hematopoietic progenitor cells.215 Wnt 3a has been shown to expand long-term repopulating HSCs.216,217 As Wnt proteins are expressed on primitive hematopoietic cells,218 it is also possible that in addition to classical paracrine signaling, Wnts could act in an autocrine fashion in HSC biology.
Transforming Growth Factor β
The transforming growth factor (TGF) family of ligands (TGF-β, activins, bone morphogenetic proteins [BMP]) bind to members of the TGF-β receptor family and trigger activation of the SMAD (Sma- and Mad-related protein) group of intracellular mediators.219 Unlike the cytokines discussed above, TGF-β members inhibit HSC cycling,220,221 and so blunt cell expansion, at least in vitro. Nevertheless, the situation in vivo is complex; genetic elimination of TGF-β does not alter HSC self-renewal or regeneration in vivo,222 likely because of redundancy in the TGF-β system of ligands.223 In contrast, genetic elimination of several of the SMAD proteins disrupts normal HSC homeostasis.224,225 Recent data suggests that BMP4 might be the critical member of the TGF family that affects HSC biology.226
The mechanisms by which these cytokines exert their effects on HSCs are only now beginning to be understood at the molecular level, but it is already clear that effects on the transcription factors that govern HSC survival, self-renewal, and expansion likely play critical roles. It has long been understood that Wnt proteins act to stimulate an increase in intracellular levels of β-catenin, a nascent transcription factor. Upon being liberated from proteasomal degradation in the presence of Wnt, β-catenin translocates to the nucleus and alters transcription of genes displaying the T-cell factor (TCF)/lymphoid-enhancer binding factor (LEF) consensus sequence.227 Moreover, TGF-β–induced alterations in SMAD protein phosphorylation affects their ability to activate transcription directly.228 However, most of the cytokine receptors that affect HSCs do not directly affect transcription factors; rather, several cytokines affect signaling pathways that alter the expression, activity, or subcellular localization of HSC transcription factors.
As discussed in “Hematopoietic Stem Cell to Common Myeloid Progenitor Commitment” above, SCL is a helix-loop-helix transcription factor critical for hematopoiesis. SCF enhances the survival of primitive hematopoietic cells in culture by maintaining their expression of SCL,131 which enhances expression of the SCF receptor c-Kit.229 Two additional transcription factors that play vital roles in HSC expansion, HOXB4 and HOXA9, are both affected by cytokines. Exogenous expression of HOXB4 to levels only twice normal are associated with a marked and rapid expansion of transduced HSCs on their transplantation into lethally irradiated recipients.116 In both model cell lines and primitive hematopoietic cells TPO doubles the expression of HOXB4, in a p38 MAPK-dependent fashion.230 Of probably greater significance is the effect of TPO on HOXA9, a gene that also induces rapid expansion of HSCs on its introduction into these cells, and whose genetic elimination leads to a profound deficit in numbers of HSC in vivo.118 Although the hormone fails to affect total cellular levels of HOXA9 in either model cells or primary primitive murine HSC populations, TPO greatly enhances HOXA9 nuclear translocation by inducing expression of its translocation partner, MEIS1, and leading to ERK1/2 MAPK-induced MEIS1 phosphorylation.231
A third mechanism by which cytokines affect HSC expansion is through global inhibitors of signaling. In addition to its direct effects on HSC survival and self-renewal pathways, TPO has been shown to interact with the adaptor protein LNK,232 which inhibits signaling pathways derived from a broad range of hematopoietic cytokines,233,234 including TPO.235 From these data it appears that TPO and LNK alternately regulate HSC expansion and each other.236
FN is a 450-kDa fibril-forming glycoprotein composed of two subunits that is a major component of the hematopoietic microenvironment. FN is produced by both marrow stromal (endothelial cells and fibroblasts) and blood cells,237 and is implicated in marrow homing of hematopoietic cells.238 Distinct domains of FN have been identified that interact with different integrins, for example, those for integrin α4β1 and for integrin α5 β1.148 HSCs display multiple integrins and their engagement contributes to cell survival and/or expansion. For example, ex vivo culture of human CD34+ cells on FN maintains the repopulating capacity of HSCs, whereas growing the cells in suspension obliterates their ability to repopulate hematopoiesis.239 FN binding to α4β1 integrins also enhances the generation of large numbers of committed hematopoietic progenitors240 and LTC-IC241 from primitive precursors. Multiple molecular mechanisms for the effects of FN on integrin-bearing cells have been identified, and serve as a paradigm for the supportive effects of this entire class of microenvironmental signals.
Integrin engagement by FN triggers a number of intracellular signaling events that affect the cellular cytoskeleton and transcriptional events. Complexes composed of kinases, adaptors, and cytoskeletal components are recruited to sites of integrin engagement, initiated by interactions with integrin cytoplasmic domains.89 A critical molecule for integrin-based signaling is paxillin, a 68-kDa protein that contains a number of protein–protein binding domains, and which binds to the cytoplasmic domain of the integrin.242 Additional binding partners also help trigger intracellular signaling, including focal adhesion kinase (FAK) and the closely related Pyk2 kinase. Upon recruitment, FAK and Pyk2 are activated and initiate Tyr phosphorylation of paxillin and other associated molecules, creating additional protein binding sites and activating tethered secondary messenger molecules. One vital signaling pathway downstream of FAK and Pyk2 is PI3K, which is mediated by the association of its regulatory p85 subunit with the adhesion kinases (Chap. 14).243 FAK also directly activates a pathway that results in upregulation of the cyclin D promoter,244 affecting cell proliferation. Integrin engagement also leads to Src activation, engagement of Grb2, and activation of Ras,245 pathways also activated by SCF and TPO, and potentially providing a mechanism by which diverse extrinsic stimuli of HSCs may converge.
Another stromal cell matrix glycoprotein is hyaluronan, which binds to two hematopoietic cell-surface receptors, RHAMM and CD44. Although most CD34+ marrow cells express CD44, only a fraction of them adhere to hyaluronan,246 a process that can be mediated by cytokines, as a result of either increased surface expression of CD44 or an alteration in its conformation. Consistent with the latter notion, certain epitopes on CD44 have been shown to be inducible,247 and antibodies to CD44 can alter the adherence of CD34+ cells to marrow stroma.248 Nevertheless, other data suggests that RHAMM is the primary receptor for hyaluronan.249 It is also of considerable interest that primitive hematopoietic cells also express hyaluronan, and that it plays an important role in their lodgment in the marrow and subsequent proliferation.250
LTCs that support hematopoiesis develop a heparan sulfate proteoglycan layer. Immunochemical analysis has shown that marrow stromal cell lines synthesize and secrete numerous members of the syndecan family of heparan sulfate, including glypican, betaglycan, and perlecan.18 Evidence is accumulating that heparan sulfate-containing proteoglycans may be vital components of the stem cell niche. For example, the structure of the heparan sulfate secreted from stromal cell lines that support long-term hematopoiesis are significantly larger and more highly sulfated than heparan sulfate from nonsupportive stromal cell lines, and when used alone in LTCs, the former can support LTC-IC, whereas desulfated heparan sulfate cannot.251
Tenascins are large, extracellular matrix (ECM) glycoproteins found in several tissues, synthesis of which is upregulated in response to tissue regeneration. Tenascins are multimeric proteins composed of numerous modules. For example, tenascin-C is composed of six subunits linked like spokes in a wheel by their C-terminal fibrinogen-like domains, each subunit being composed of multiple epidermal growth factor (EGF)-like and FN type III modules. Two forms of tenascin of molecular mass (Mr) 280 and 220 kDa are also expressed at high levels by marrow stromal cells.252 Marrow cells can adhere to tenascin-C within the fibrinogen-like domain and to two sets of the FN type III-like repeats, and when so engaged, they undergo a proliferative response.253 Genetic elimination of tenascin leads to modest deficiencies in marrow hematopoietic progenitor cells,254 although as the levels of FN in such mice are also reduced, it is unclear if direct tenascin engagement of hematopoietic cells is responsible, or the defect is a result of the secondary reduction of FN engagement of β1 integrins.
Laminins are heterotrimeric (αβγ) extracellular proteins that regulate cellular function by adhesion to integrin and nonintegrin receptors. At present, 5 α chains, 3 β chains, and 2 γ chains have been characterized, which combine to form at least 12 distinct laminin isoforms.255 Laminins containing γ2 and either β1 and α5 chains are expressed in marrow, but only the latter (laminin-10/11) binds to α6 β1 integrin on primitive hematopoietic cell lines256 and to primary human CD34+/CD38− stem and progenitor cells.257 A second, nonintegrin laminin receptor (LR) also binds laminins, as well as other components of the ECM, such as FN, collagen, and elastin, and is composed of an acylated dimer of 32-kDa subunits.258 Although not an integrin, the LR associates with integrins (e.g., integrin α6 β4) to modulate laminin binding.259 Functionally, aminin-10/11 facilitates SDF-1α–stimulated transmigration of CD34+ cells,260 and displays mitogenic activity toward human hematopoietic progenitor cells.255 The nonintegrin LR associates with the GM-CSF receptor (GM-CSF-R) to modulate its signaling properties, down-modulating receptor signaling in the absence of laminin, and releasing the inhibition when bound by its ligand.261 This arrangement could provide a novel molecular explanation for how laminins affect cell proliferation; whether this physiology extends to other cytokines that affect HSCs is under investigation.
Collagen Types I, III, V, and VI
Collagen types I, III, IV, and VI have been identified in LTC or in situ from marrow sections by a number of methods.35,262 Most of the marrow-derived collagen types are assembled into long fibrils, which form the fine, background reticulin staining seen on marrow biopsies, although type IV collagen is assembled into a meshwork seen most commonly as part of basement membranes. Collagens also interact with laminins in the marrow. Collagen types I and VI are strong adhesive substrates for various hematopoietic cell lines and marrow mononuclear cells, including committed myeloid and erythroid progenitors.262 Classic collagen receptors on blood cells are of two types, the β1 integrins (α1β1 and α2β1) and the nonintegrin glycoprotein VI, present predominantly on platelets.
THE AGING MARROW MICROENVIRONMENT
Like the HSC itself (see “Stem Cell Aging” earlier), the HSC niche undergoes several changes with aging. Although the number of osteoblasts decreases, they generate higher levels of ROS, inducing p38 MAPK signaling, potentially accounting for the reduction in self-renewal capacity of HSCs derived from older mammals. The number of adipocytes increases as a result of the skewed differentiation of aged MSCs; increased adiposity and reduced osteogenesis lead to decreased CXCL12 levels in the aged marrow. This finding could be responsible for altered HSC mobilization in elderly individuals. In contrast, increased levels of the CC-chemokine ligand 5 (CCL5; also known as RANTES [regulated upon activation, normal T-cell expressed and secreted]), in the niche could contribute to the altered myeloid/lymphoid skewing seen in HSCs of older individuals.263 This topic has been reviewed but clearly requires additional study.138
CONTROVERSIES IN HEMATOPOIESIS
LINEAGE FATE DETERMINATION
One of the most contentious issues in hematopoiesis is the origin of stem cell commitment to specific blood cell lineages. Two schools of thought exist: extrinsic and intrinsic control. The former, championed by Metcalf and others,264 argues that cytokines, ECM, or other stimuli instruct the hematopoietic stem or progenitor cell to differentiate into specific cell types. In contrast, Dexter and others265 argue that a hierarchy of transcription factors direct a cell toward a specific lineage, mechanistically explained by a stochastic rise in one or more of a mutually antagonistic set of transcription factors, that drive developmental pathways by enhancing expression of the genes that characterize that pathway, and by interfering with the levels or function of the transcription factors that drive the alternate lineage fate choice.
The Case for Transcription Factors
A strong case has been made for intrinsic control of stem cell lineage determination.265 As Enver and colleagues state: “Simply put, the question is this: Is unilineage commitment the result of a cell-autonomous, internally driven program, or rather is it the consequence of a cell responding to an external, environmentally imposed agenda?” These and several other investigators argue that the stochastic rise in one or another lineage determining transcription factor in the multilineage progenitor leads to its ultimate lineage commitment.
It is abundantly clear that transcription factors can direct lineage commitment in hematopoietic cells. A partial list of transcription factors restricted to specific hematopoietic lineages includes Pax5 (B cells),266 Ikaros (B/T cells),267 PU.1 and C/EBPα (myeloid and B cells),268,269 GATA1 (erythrocytes and MKs),132,270 Fli1 (MKs),271 and C/EBPε (granulocytes).272 A number of loss-of-function studies have revealed the nonredundant role of these proteins in development of the corresponding cell lineage. For example, genetic elimination of Pax5 eliminates B cells273; elimination of Ikaros leaves a mouse devoid of fetal T cells, fetal and adult B cells, and their progenitors124; and loss of C/EBPα leads to absolute neutropenia.274 Moreover, the exogenous expression of several transcription factors in lineage committed progenitor cells can redirect cell fate. For example, C/EBPα is expressed in myeloid progenitor cells, and introduction of a regulatable C/EBPα gene into purified erythroid progenitors causes their switch to the myeloid lineage.275 In further support of this hypothesis, several lines of evidence have been gathered, including the finding that forced expression of the antiapoptotic gene bc12 in a growth factor-dependent multipotential hematopoietic cell line resulted in growth factor independence and spontaneous differentiation into all of the possible cell lineages that develop when the corresponding growth factor(s) are added to the wild-type cells.276
In addition to providing these and other arguments in favor of a transcription factor–based intrinsic regulatory mechanism of stem cell fate, proponents of the intrinsic hypothesis point to feed-forward switch-like molecular mechanisms in which a stochastic increase in one of a binary set of such transcription factors reduces the level or activity of those transcription factors responsible for alternate cell fates. An example of this physiology is illustrated by the mutually antagonistic effects of the erythroid transcription factor GATA1 and the myeloid transcription factor PU.1; GATA1 acts to inhibit the myeloid activation potential of PU.1,277 and PU.1 blocks the binding of GATA1 to its genetic target sites.278 Thus, when the level of GATA1 stochastically rises above that of PU.1 in a CMP, the granulocyte-macrophage potential would be extinguished and the MEP potential of the cell would march forward, unfettered. Alternately, CMPs in which PU.1 levels rise above that of GATA1 would develop along the myeloid lineages, both through the direct stimulation of myeloid gene expression by PU.1, and indirectly by the blockade of GATA1-mediated erythroid and megakaryocytic gene expression programs.
The Case for Humoral Mediators
Although much evidence has been garnered in favor of an intrinsic mechanism of stem and progenitor cell fate determination, proponents of an extrinsic instructive hypothesis have also generated a large amount of compelling evidence in favor of the importance of extrinsic signals. One illustrative example of the capacity of certain extrinsic signals to impact specific patterns of differentiation is that the exogenous expression of an IL-2Rβ transgene in CLPs induces their differentiation into myeloid cells.279 Subsequent studies revealed that the presence of the exogenous receptor leads to upregulation of the GM-CSF-R in the CLP, and that exogenous expression of GM-CSF-R could also lead a CLP toward monocyte/macrophage development.2 In separate studies, other cytokines were shown to direct myeloid lineage fate determination; compared to the differentiation profile seen when marrow cells were cultured with SCF alone, an antiapoptotic stimulus, the addition of IL-5 greatly enhanced the number of marrow progenitor cells that gave rise to eosinophilic colonies, whereas the addition of TPO induced a predominance of megakaryocytic colonies, without significant changes in the number of apoptotic cells in any of the three culture conditions. These results were interpreted to indicate that while the SCF could keep nearly all progenitor cells alive under the cell culture conditions employed, the second cytokine directed the multilineage progenitors into specific cell fates.280
A number of external signaling events have been found to directly impact the transcriptional apparatus of the cell. For example, as previously noted, two transcription factors that lead to the self-renewal and expansion of HSCs, HOXB4, and HOXA9 are induced to higher levels of expression or to translocate into the nucleus of stem cells in response to TPO.235,236 Moreover, SCL, a transcription factor that when expressed in maturing hematopoietic cells inhibits cytokine-induced granulocytic and monocytic differentiation, maintaining them in an undifferentiated state, is enhanced by SCF and down-modulated by GM-CSF.131 And the level of c-Myb, which determines whether a MEP develops an erythroid or MK fate is affected by TPO, mediated by its induction of miR150.281 Thus, strong evidence supporting both extrinsic and intrinsic control of lineage determination has been presented, and like the case for most conflicts in biology, it is most likely that elements of both mechanisms operate in hematopoiesis.