Hematopoietic Stem Cells
9.2 The Hematopoietic Lineage Commitment Process
The generation of a fully functional lympho-hematopoietic system is a gradual yet dynamic process. As the differentiation-committed progeny of LT-HSCs expand, they undergo progressive gene-expression changes that ultimately result in the development of mature cells with a range of very different func-tional capacities. In hematopoiesis, the process of differentiation is tightly coupled with proliferation.
Progenitor cell subtypes and HSCs are morphologically indistinguishable, unlike precursors and mature cells. Therefore, we rely on immunophenotyping schemes to identify and purify cell subsets enriched in each population specified by hematopoietic models. Characterization of distinct cell popula-tions by stage and lineage is achieved best by cell surface marker stains, using monoclonal antibodies, and/or dye efflux capacity (discussed later in detail) and assessed by fluorescence-activated cell sorting (FACS), followed by assays to assess their functional capacity. However, given the fluid, gradual nature of differentiation, a cell population that we might define operationally as representative of a particular stem-progenitor subtype, actually is a snapshot of cells at approximately that stage and lineage of hemato-poietic differentiation. In addition, an immunophenotypically defined population has a high probability of containing slightly varied types of cells with respect to proliferative state, as well as stage and lineage.
Thus, we must emphasize that a cell type of interest can rarely be “purified” to complete homogeneity;
instead, the term “enriched” better describes the limited heterogeneity of any FACS-sorted population.
Currently, the accepted model of hematopoiesis (Figure 9.2) is a schematic representation of a fluid process. Each hematopoietic stem or progenitor cell (HSPC) subtype is an ancestral cell that is defined by the types of mature cells that it can produce and the duration of time over which it can sustain mature cell production. The dynamic nature of the differentiation process implies the “Heisenberg”-type impossibility of simultaneously isolating a single cell for some novel analysis and functionally defining the same cell and its progeny in a functional assay. These progenitor populations should therefore be treated as our best approximation or model of the process of hematopoiesis, rather than as actual cells.
Therefore, we should remain open to the possibility, for example, that an enriched subset of “myeloid”
or “lymphoid” progenitors might have the capacity to give rise to other lineages under appropriate con-ditions. A recent study illustrates the ability of human monocytes and dendritic cells to arise from a population previously believed to be lymphoid-restricted (Doulatov et al. 2010).
The following section will describe the process of lineage commitment in hematopoiesis, according to established models.
9.2.1 Common Myeloid Progenitors and Their Progeny
The current model of hematopoiesis proposes the existence of a CMP, a myelo-erythroid restricted pro-genitor. The CMP proliferates and gives rise to two bi-potent progenitors, the granulocyte−monocyte progenitor (GMP) and the megakaryocyte−erythroid progenitor (MEP).
9-4 Tissue Engineering
9.2.1.1 Red Blood Cells (Erythrocytes)
Mature red blood cells, or erythrocytes, deliver oxygen to the tissues. They are rich in cytoplasmic hemoglobin, an iron-containing protein responsible for their red color, which binds oxygen with a high affinity. Erythrocytes develop from erythroid-committed progenitors within the bone marrow in a pro-cess termed erythropoiesis. In semisolid cultures containing erythropoietin (EPO), early and late ery-throid progenitors are characterized functionally as BFU-E (burst-forming units eryery-throid) and CFU-E (colony-forming units erythroid), respectively; the names of erythroid colonies have been preserved over their long history. Erythroid-committed progenitors express glycophorin A (CD235a) and high levels of transferrin receptor (CD71) (Loken et al. 1987, Socolovsky et al. 2001). Expression of glycopho-rin A is erythroid-specific. Although essentially every mitotic cell type expresses transferglycopho-rin receptor, because iron is required for cell division, erythroid progenitors can be distinguished by their very high CD71 expression, consistent with the high levels of hemoglobin being synthesized. As they differenti-ate, erythroid precursors undergo sequential morphological changes before extruding their nuclei and becoming reticulocytes, which in turn develop into mature erythrocytes that circulate for 3 months in humans. Senescent erythrocytes are endocytosed by macrophages which recycle their iron back into the circulation (Koury et al. 2002).
9.2.1.2 Platelets
Platelets are anucleate cell fragments that bind to damaged tissues where they are instrumental in the blood clotting and wound-healing processes. Megakaryoblasts and megakaryocytes are multinucleated
Megakaryocytes Erythrocytes Granulocytes
Monocytes
T cells NK cells B cells Dendritic
cells LT-HSC Long-term HSC
ST-HSC Short-term HSC
MPP Multipotent progenitor
Common myeloid progenitorCMP
GMP Granulocyte-monocyte progenitor Megakaryocyte-MEP
erythroid progenitor
CLP Common lymphoid progenitor
ProB
ProT NKP
FIGURE 9.2 Tree diagram of hematopoiesis. Hematopoiesis is organized in a hierarchy that is sustained by a very small population of quiescent, LT-HSCs that persist for the lifetime of the organism. These cells are capable of differ-entiating sequentially into ST-HSCs and MPPs that have reduced self-renewal capacity but greater proliferation. The progeny of MPPs become lineage-restricted CLPs or CMPs. Their progeny gradually expand and differentiate into vast numbers of morphologically-recognizable precursors and then mature functional blood and immune cells, many of which are non-proliferative and have short half-lives. An exquisitely regulated balance between the self-renewal and the differentiation of HSCs allows for a lifetime of blood cell production, without exhausting the small pool of HSCs.
platelet precursors that develop from MEPs. Megakaryocytic progenitors will form CFC-Mk in meth-ylcellulose in the presence of thrombopoietin (TPO). Platelets and megakaryocytes can be immu-nophenotyped based on their expression of cell surface CD41, CD42a, CD42b, CD42c, CD42d, and CD61— receptors for the clotting factors fibrinogen, fibronectin, and von Willebrand factor (Kaushansky 2008). Mature megakaryocytes migrate to capillary sinusoids where they shed mature platelets into the circulation. A single megakaryocyte can produce over 1000 mature platelets (Kaushansky 2008) which circulate for around 10 days in the human body (Mason et al. 2007).
9.2.1.3 Granulocytic and Monocytic Cells
Precursors of granulocytes and monocytes arise from GMPs. The close developmental relationship between granulocytes and monocytes can be demonstrated simply in a colony-forming assay. Whole bone marrow plated in semisolid medium containing IL-3, IL-6, and SCF for mouse or IL-3, SCF, G-CSF and GM-CSF for human, readily forms bipotent CFC-GM, which contain both granulocytes and mono-cytes, as well as unipotent CFC-G and CFC-M, which form only granulocytes or monomono-cytes, respec-tively. CD33 is a pan myeloid marker. Human monocytes and granulocytes can be distinguished by their expression of CD14 (Goyert et al. 1988) and CD15 (Skubitz et al. 1988), respectively. In the mouse, monocytes can be identified with CD115 (the M-CSF receptor) and granulocytes by their high Gr1 expression (Alder et al. 2008). Developing granulocytes can be identified morphologically at the pro-myelocyte stage when their cytoplasmic granules become evident in microscopic evaluation of Wright’s stained histologic bone marrow cell smears. Mature granulocytes are easily identifiable morphologi-cally due to their highly compacted nuclei that become multilobed, in human, or ring shaped, in mouse (Friedman 2002). Neutrophils, the major type of granulocytes, are the shortest-lived mature cell type in the hematopoietic system with a half-life on the order of days (Pillay et al. 2010). Neutrophil gran-ules are loaded with potent enzymes and reactive oxygen species (ROS) that function as a first line of defense against invading pathogens. Each day, about 109 neutrophils/kg of body weight, in the human, die via apoptosis and are efficiently phagocytosed by monocytes and macrophages (Luo and Loison 2008), which prevents the potentially harmful release of their enzymes and ROS. Monocytes are phago-cytic cells that engulf pathogens and cellular debris that they encounter. Monocytes develop into tissue macrophages and antigen-presenting dendritic cells.
9.2.2 Common Lymphoid Progenitors and Their Progeny
The current hematopoietic model also proposes the existence of a lymphoid-restricted CLP, which can be separated from the CMP based on expression of the IL-7 receptor. The progeny of the CLP develop into B cell precursors that remain in the bone marrow or T cell precursors that migrate to the thymus.
Although mature lymphocytes are generally quiescent, specific memory T and B cells can be reactivated by their cognate antigen to proliferate, expand, and fight infection.
9.2.2.1 B Cells
B cells are effector cells of the adaptive immune system producing surface bound immunoglobulins (antibodies) that can be secreted upon B cell activation and differentiation into plasma cells. B cells acquired their name because their development in birds takes place in a specialized organ called the bursa of Fabricius. The bursa of Fabricius is absent in mammals, and B cell development proceeds in the bone marrow, in close proximity with specialized stromal cells, where each cell rearranges the genetic regions responsible for antibody-binding specificity, called variable regions. This process occurs by ran-domly joining gene segments, in a process called V-D-J rearrangement, and introducing mutations at the junction regions to produce a repertoire of B cell clones with a vast variety of different variable regions.
Those B cells that make productive rearrangements, such that their entire immunoglobulin proteins are intact, are positively selected for and survive; those that do not make productive rearrangements do not receive positive signals and undergo apoptosis. Finally, B cells with productive rearrangements that
9-6 Tissue Engineering have high affinity for self-proteins are eliminated by a process called negative selection, which provides protection against deleterious autoimmune reactions (LeBien and Tedder 2008).
B cells that encounter a molecule (usually a protein) that binds to their specific immunoglobulin, called an antigen, can undergo further affinity maturation in secondary lymphoid organs such as the spleen or lymph nodes. In this process, activated B cells proliferate and acquire additional genetic changes in their immunoglobulin variable regions, a process called somatic hypermutation, to give rise to subclones produc-ing antibodies with even greater affinity for that antigen. The expansion of activated B cell clones and their affinity maturation is the basis for the accelerated secondary response to infections that an individual has previously encountered. B cells have extremely long half-lives and can persist in the circulation or tissues for years to decades (Matthias and Rolink 2005). Early developing B cells express CD19 and CD10. Mature B cells can be distinguished by their expression of CD21 and CD22 and the lack of CD10 (Loken et al. 1988).
9.2.2.2 T Cells
The process of T cell development is analogous to B cell development, although T cell development takes place in the thymus. Developing thymocytes rearrange the genetic regions that encode their T cell receptors. There are two major differences between B cell receptors—surface bound antibodies—and T cell receptors. B cell receptors can be surface bound and then secreted, following activation, whereas T cell receptors are always surface bound. B cell receptors bind directly to antigens while T cell receptors can only recognize peptide antigens expressed by MHC molecules. There are two classes of MHC pro-teins. MHC class I molecules are expressed by all nucleated cells, and present peptides from within the cell. Thus, the peptides expressed on MHC class I represent a random sampling of the proteins within a given cell. These peptides can be nonimmunogenic portions of self-proteins or degraded from an invading pathogen or altered self-protein (e.g., in a viral infection or cancer, respectively). MHC class II molecules are expressed only by professional antigen-presenting cells (APCs)—dendritic cells, activated B cells or activated macrophages—and are “loaded” with MHC-bound peptides from proteins that have been phagocytosed by these APCs.
Early thymic progenitors (ETP) have migrated from the bone marrow and retain the ability to dif-ferentiate into monocytes, NK cells, and dendritic cells. ETPs begin at the double negative (DN) stage, as they do not express CD4 or CD8, co-receptors for the TCR. DN progenitors gradually rearrange their TCR genes. A productive rearrangement results in positive selection and progression to the double posi-tive (DP) stage, where both CD4 and CD8 are expressed. DP progenitors successively undergo negaposi-tive selection, in order to eliminate any T cell clones with high affinity for self-peptides expressed by MHC molecules on thymic stromal cells, followed by the fate decision to either CD4+ or CD8+ expression.
T cell clones will also expand upon activation by binding to an MHC molecule and peptide combina-tion for which their TCR and co-receptor will bind. CD8+ “cytotoxic” T cells eliminate host cells that express a peptide−MHCI combination that a given T cell clone recognizes as foreign. CD4+ “helper” T cells play an instrumental role in activating B cells that express a peptide−MHCII combination that they recognize. Similarly to B cells, T cells are extremely long-lived (Rothenberg et al. 2008). Mature T cells can be identified by their expression of CD3, CD4 or CD8, and TCR (Toribio et al. 1988).
9.2.2.3 Dendritic Cells
Dendritic cells (DCs) are the most potent (therefore, called “professional”) APCs: their role in the immune system is to constantly phagocytose antigens and display their peptides on MHC molecules.
DCs can be found in the spleen, skin, lymph nodes, lung, liver, and kidney. Tissue-resident DCs can have very long half-lives.
There are a variety of tissue resident DC types, each with specific cell surface markers and gene-expres-sion profiles. For example, in the spleen there are both CD8+CD205+ and CD8−33D1+ DC sub-types. The CD8+ DCs in the spleen are more efficient at phagocytosing apoptotic bodies and “cross-priming” the antigens onto MHC class I molecules, for the detection of viral and tumor antigens. The CD8− subtype presents phagocytosed antigens on MHC class II molecules in the more classical method.
The origin of DCs is somewhat promiscuous, and both highly purified CMPs and CLPs have the capacity to generate DCs. In the bone marrow, DCs can be traced back to a bipotent monocyte-DC progenitor (MDP). ETPs and early DN T cell precursors also have the capacity to generate DCs (Liu and Nussenzweig 2010, Rothenberg et al. 2008).
9.2.2.4 Natural Killer Cells
Natural Killer (NK) cells are lymphocytes that are instrumental in the cellular response against tumors and pathogen-infected cells. Mature NK cells express cell surface receptors that bind to MHC class I molecules on the surface of host cells. Viral infections and tumor initiation are often accompanied by aberrant expression of MHC class I molecules, which can be recognized by these receptors, and thereby trigger NK cells to release cytotoxic proteins (perforins and granzymes) from their granules.
Additionally, NK cells can be activated to release the contents of their granules upon recognition of antibodies bound to the surface of a pathogen infected cell, in a process called antibody-dependent cel-lular cytotoxicity (ADCC).
NK cell development proceeds from the CLP stage in the bone marrow and from ETPs in the thymus, but these two populations of NK cells may be functionally distinct. Unlike other lymphocyte types, NK cell development does not require the rearrangement of any specialized receptors. NK precursors in the bone marrow express CD122, the common β chain of the IL-2 and IL-15 receptors. As NK precursors differentiate, they gradually acquire the expression of cell surface molecules required for their function including CD56, CD94 (MHCI binding protein) and CD16/CD32 (FcRγ) (Boos et al. 2008).
9.2.3 Multipotent Cells
The most primitive hematopoietic cells—long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), and multipotent progenitors (MPPs)—are categorized functionally based on the duration for which they can support multilineage blood production. MPPs and ST-HSCs provide short-term reconstitution to a transplant recipient for up to 4–6 weeks and 3–4 months (Yang et al. 2005), respectively. A true LT-HSC, at the apex of the hematopoietic hierarchy, is capable of providing an entire lympho-hematopoietic system to a transplant recipient for a lifetime (Osawa et al. 1996) and can do this repeatedly as can be revealed by serial transplantation into secondary, tertiary, and subsequent recipients. While, all of these multipotent cell types satisfy one requirement for defining stem cells, the capacity for multilineage dif-ferentiation, only the LT-HSC has the capacity for such extensive self-renewal. Thus, the most rigorous test for a stem cell is self-renewal, as assessed by the serial transplant assay. For the purpose of this chap-ter, we will refer to human LT-HSCs simply as HSCs, since they have not been fractionated to the same purity as mouse LT-HSCs, mainly due to both the paucity of markers available to characterize human HSCs, and suboptimal immunodeficient mouse models (discussed below in detail).
9.3 Hematopoietic Stem Cells
LT-HSCs are extremely rare, comprising <0.005% (based on our own observations and Kiel et al. 2005, Yang et al. 2005) of nucleated cells in adult mouse bone marrow. The remaining > 99.99% of the marrow is comprised of the progeny of LT-HSCs.
9.3.1 Identification Strategies
It is important to remember that there is no single marker or characteristic that perfectly identifies HSCs.
Obtaining HSC preparations of absolute purity proves to be challenging, and as mentioned above, most studies achieve “enrichment” rather than “isolation” of HSCs or other rare hematopoietic subsets. In addition, HSCs and other hematopoietic subsets must be defined by function. Morphologically, HSCs are virtually indistinguishable from lymphocytes. Indeed, the first enrichment strategies relied on the
9-8 Tissue Engineering observation that HSCs are small cells. Using the CFU-S assay, which was believed to be an HSC assay at the time, fractions of small cells that had been separated by density-gradient sedimentation were found to be enriched in CFU-S (Worton et al. 1969). Later it was shown that HSCs could be fractionated away from CFU-S by elutriation, which separates cells based on their size and density (Jones et al. 1990).
Currently, more sensitive methods are available to identify HSCs.
9.3.1.1 Immunophenotype
Using FACS, subsets of cells can be prospectively enriched, based on their light scattering characteris-tics and cell surface marker expression then probed for their ability to reconstitute the hematopoietic system. Simple immunophenotypic cell purifications can also be performed utilizing magnetic beads instead of FACS. Magnetic separations are robust and cost effective, and therefore can be used clinically (in the case of CD34 for human hematopoietic stem-progenitor cells (HSPCs) (Civin et al. 1996)) or to partially purify cell subsets prior to cell sorting for definitive cell purification.
9.3.1.1.1 Enriching for Mouse HSCs
HSPCs in the mouse are found within the Kit+Sca+Lin− (KSL) population (Orlic et al. 1993, Spangrude et al. 1988) which comprises ~0.25% of whole bone marrow cells. This population includes LT-HSCs, ST-HSCs and MPPs. One in 30 KSL cells generates long-term engraftment in an irradiated recipient mouse. The KSL population can be further subdivided using either of two marker schemes, either the KSLCD34loFlt3− LT-HSC definition (Yang et al. 2005), the KSLCD150+CD48− definition (Kiel et al.
2005), or a combination of the two to achieve extremely high purity (requiring seven-color flow cytom-etry) (Wilson et al. 2008).
9.3.3.1.2 Enriching for Human HSPCs
Human HSPCs can be isolated from bone marrow, umbilical cord blood or mobilized peripheral blood (see sources of HSCs below). In vivo repopulating HSCs are enriched in the CD34+CD38− population (Civin et al. 1984, 1987, Terstappen et al. 1991). This population is approximately equivalent in purity to the mouse KSL population—it is enriched for HSCs but ~95% are progenitors and will not provide long-term hematopoiesis. Somewhat enhanced purity can be achieved using CD90 (Thy1) and CD45RA; LT-HSCs are enriched in the CD34+CD38−CD90+CD45RA− cell population (Baum et al. 1992, Majeti et al. 2007).
9.3.1.2 Functional Characteristics 9.3.1.2.1 Side Population
Goodell and colleagues first demonstrated that HSCs could be enriched based on efflux of the DNA staining dye Hoechst 33342 (Goodell et al. 1996). Mouse bone marrow stained with Hoechst 33342 at 37°C displays a curious staining pattern when visualized on a flow cytometer. A small subset of cells, known as the side-population (SP), can be observed with low blue and red fluorescence. The SP subset can be abolished by incubating the cells with Verapamil or other MDR (multidrug resistance) pump inhibitors. The SP technique can be combined with immunophenotypic markers to more highly enrich for HSCs (Weksberg et al. 2008).
Goodell and colleagues first demonstrated that HSCs could be enriched based on efflux of the DNA staining dye Hoechst 33342 (Goodell et al. 1996). Mouse bone marrow stained with Hoechst 33342 at 37°C displays a curious staining pattern when visualized on a flow cytometer. A small subset of cells, known as the side-population (SP), can be observed with low blue and red fluorescence. The SP subset can be abolished by incubating the cells with Verapamil or other MDR (multidrug resistance) pump inhibitors. The SP technique can be combined with immunophenotypic markers to more highly enrich for HSCs (Weksberg et al. 2008).