Stem cells

Assays for stem cells

Stem cells are found at a very low frequency in haemopoietic tissue and cannot be recognized in stained smears of bone marrow. In animal models, the best assay for stem cells is a repopulating assay, which tests the ability of the cells to engraft and restore haemopoiesis in a myeloablated host. For human stem cells, a variety of in vitro assay systems have been used, but the most widely accepted is the long-term bone marrow culture system (LT-BMC). This method reproduces the microenvironment of the bone marrow in vitro by growing a feeder layer of stromal cells. When haemopoietic long-term culture-initiating cells (LT-CICs) are seeded onto the stromal layer, they are induced to proliferate and produce progenitor cells that can be measured in the clonogenic assays detailed below. However, the endpoint of this assay is indirect, in that committed colony-forming cells produced by the LT-CICs are enumerated. Consequently, limiting dilution analysis is necessary to determine LT-CIC numbers. However, limiting dilution is statistically based and the procedure is very cumbersome.

A variety of clonal assays for candidate stem cells have also been used. The cobblestone area-forming cell (CAFC) assay resembles the long-term culture assay in that haemopoietic cells are seeded onto stromal layers. In this case, however, the formation of colonies containing cells of a cobblestone-like morphology is observed. The blast colony-forming cell (Bl-CFC) assay and high proliferative potential colony-forming cell assay (HPP-CFC) detect cells that share certain characteristics with stem cells but are generally considered to be less primitive than stem cells.

Pluripotent stem cell

Pluripotent stem cell

CFU-GEMM

Lymphoid stem cell

CFU-Mk

CFU-E

Erythrocytes Neutrophils Monocytes/ Eosinophils Megakaryocytes/

macrophages platelets

CFU-E

CFU-G CFU-M

Erythrocytes Neutrophils Monocytes/ Eosinophils Megakaryocytes/

macrophages platelets

CFU-Mk

Figure 1.3 Hierarchical organization of haemopoiesis.

Stem cell properties, phenotype and purification

Morphologically, haemopoietic stem cells are undifferentiated and resemble small lymphocytes. Normally, a large fraction is quiescent, in G0 phase of the cell cycle, which protects them from the action of cell cycle-dependent drugs such as 5'-fluorouracil, and S-phase-specific agents such as cytosine arabinoside and hydroxyurea. The quiescent state of stem cells is maintained by transforming growth factor P (TGF-P). The activity of TGF-P is mediated by p53, a tumour suppressor that regulates cell proliferation and targets the cyclin-dependent kinase inhibitor p21. The cyclin-dependent kinase inhibitors regulate the activities of cyclin-cyclin-dependent kinase (CDK) complexes. Inhibition of the cyclin-CDK complexes prevents phosphorylation of the retinoblastoma proteins that remain bound to transcription factors belonging to the E2F family. As a consequence, genes required for progression of the cell cycle are not transcribed and cells remain quiescent (Figure 1.4). Stromal cells express TGF-P, which is involved in maintaining stem cell quiescence in the bone marrow microenvironment.

The immunophenotype of haemopoietic stem cells is summarized in Table 1.1 and consists of the presence of markers that are expressed by stem cells (CD34, Thy-1) and those that are absent (CD33, CD38, HLA-DR and lineage-specific (lin) markers). CD34 is the best-known marker of human stem and progenitor cells. It is a member of the sialomucin family of glycoproteins, which are heavily glycosylated molecules with potential adhesion and signalling capabilities. CD34 has been

Figure 1.4 Maintenance of stem cell quiescence.

implicated in the binding together of cells from the KG1a line and of primary human CD34+ progenitor cells.

The importance, interest and rarity of stem cells have led to extensive efforts to purify them, based on stem cell characteristics that distinguish them from other cells in the haemopoietic

Table 1.1 Basic immunophenotype ofhaemopoietic stem cells.

Positive

Negative

CD34

CD33

Thy-1

CD38

AC133

Lineage markers

c-Kit

HLA-DR

system. The resistance of stem cells to cell cycle-dependent drugs, particularly to 5'-fluorouracil, has been used as one of the stages in stem cell purification. Fluorescence-activated cell sorting of cells labelled by monoclonal antibodies to phenotypic markers is a widely used strategy for stem cell purification, as is magnetic bead sorting. Despite efforts to purify stem cells to phenotypic homogeneity, the resulting populations are not functionally homogeneous.

Stem cell renewal and differentiation

Stem cells are capable of self-renewal and differentiation when they divide and are responsible for producing all the mature blood cells throughout life. This means that when steady-state stem cells divide, only 50% of the daughter cells, on average, differentiate, the remaining 50% do not differentiate, but maintain stem cell numbers. This could be accomplished by asymmetric cell division, so that each dividing stem cell forms one new stem cell and one differentiated cell (Figure 1.5a). Alternatively, balanced numbers of stem cells could divide symmetrically to form either two new stem cells or two differentiated cells (Figure 1.5b). Clearly, the asymmetric model does not allow for regeneration of the stem cell population but, by altering the proportions of renewing and differentiating stem cells, the symmetric division model can account for stem cell recovery (Figure 1.5c) because it permits an increase in the proportions of symmetrical self-renewing divisions and a reduction in the proportion of differentiating divisions. The symmetrical model of stem cell division means that self-renewal and differentiation are likely to be properties of the stem cell population at large, rather

Figure 1.5 Models of stem cell self-renewal and differentiation.

than characteristics of each individual stem cell. However, this mechanism is accompanied by extinction of the differentiating stem cells because the clones they produce will not contain any stem cells.

Regulation of self-renewal

Control of haemopoietic stem cell proliferation kinetics is critically important for the regulation of haemopoietic cell production. Nonetheless, information about the control of stem cell renewal versus differentiation, and how this might be manipulated to improve haemopoietic cell regeneration, is still incomplete. Control mechanisms could be intrinsic or extrinsic to the stem cells, or a combination of both.

Extrinsic factors

Extrinsic control would mean that self-renewal and differentiation can be controlled by external factors, such as cell-cell interactions in the haemopoietic microenvironment or cytokines, and thereby be responsive to demands for increased haemopoietic cell production. Regulation in the bone marrow microenvironment, and in the stromal layers of long-term bone marrow cultures, may be mediated by adjacent cells or local cytokine production. SCF is produced by stromal cells and occurs as a transmembrane protein as well as a soluble protein. It binds to its receptor, c-Kit, expressed by haemopoietic stem cells and is essential for normal blood cell production. Flt3 ligand is also a transmembrane protein and is widely expressed in human tissues. It binds to Flt3 on haemopoietic cells and is important for cell survival and cytokine responsiveness. TGF-P reduces stem cell cycling and maintains stem cell multipotency.

The Notch-1-Jagged pathway may serve to integrate extracellular signals with intracellular signalling and cell cycle control. Notch-1 is a surface receptor on haemopoietic stem cells that binds to its ligand, Jagged, on stromal cells. This results in cleavage of the cytoplasmic portion of Notch-1, which can then act as a transcription factor. c-Kit, the receptor for SCF, and receptors for TGF-P and tumour necrosis factor a (TNF-a) may also act in this way.

Intrinsic factors

The expression of several transcription factors has been shown to be essential for haemopoietic cell development from the earliest stages. SCL (stem cell leukaemia haemopoietic transcription factor) and GATA-2 are required for the development of haemopoiesis in the yolk sac, whereas absence of AML-1 results in failure of fetal liver haemopoiesis, although erythropoiesis in the yolk sac is not affected. Candidate genes that are targeted by these transcription factors include c-Kit, the receptor for granulocyte colony-stimulating factor (G-CSF), globin genes and myeloperoxidase.

There is evidence from murine studies that haemopoietic progenitor cells from different inbred mouse strains vary widely in number and proliferative activity. These observations indicate that genetically determined constitutional variation in human haemopoiesis is also likely to exist. This view is supported by the fact that parameters such as clonogenic cell frequency and numbers, proliferation ability and capacities for mobilization and expansion vary widely among individuals in the general human population. Associations have been reported between genetic markers and the frequency and activity of stem cells in mouse strains. De Haan and colleagues (2002) concluded that the expression levels of a large number of genes may be responsible for controlling stem cell behaviour. These collections of genes may be analogous to those responsible for the interindividual behaviour of human haemopoietic stem cells.

In contrast to the genetic basis for constitutional variation, certain specific genes have been demonstrated to influence haemopoietic cell kinetics. Growing evidence implicates gene products involved in cell cycle control, such as the cyclin-dependent kinase inhibitors (CKIs) p16, p21 and p27 (Figure 1.4) and the maintenance of stem cell quiescence. They have been shown to enhance proliferation and repopulating efficiency of bone marrow cells in gene knockout, knockin and gene transfer models. Loss of CKIs increases clonal expansion by haemopoietic progenitor cells and the size of the stem cell pool; the Fas and Fas ligand genes, which generally are associated with the process of cell death by apoptosis, also influence haemopoiesis as part of a mechanism suppressing progenitor cell proliferation.

Finally, lessons can be learned from studies of disease pathogenesis. Many cell cycle control genes and genes promoting cell death by apoptosis are tumour-suppressor genes that have been found to be deleted or mutated in leukaemia and other cancers. Fanconi's anaemia is an autosomal recessive bone marrow failure syndrome associated with an increased tendency for spontaneous chromosome breaks. The disease can be caused by mutations in at least seven different genes. The genes FANCA, FANCC, FANCD1, FANCD2, FANCE and FANCF have been cloned, and the corresponding proteins play important roles in DNA repair. In dyskeratosis congenita mutations have been identified in the DKC1 gene, which encodes dyskerin. Dyskerin is a component of small nucleolar ribonuclear protein particles and the telomerase complex, indicating that the disease is due to defective telomerase.

Overall, intrinsic and extrinsic control mechanisms may be considered separately, but a picture is emerging of the integration of extracellular signalling, signal transduction, transcription factors and cell cycle control in the determination of stem cell fate.

Stem cell lineage selection

The 50% of daughter stem cells that differentiate supply cells that are destined to form all of the eight blood cell lineages. The mechanisms determining the blood cell lineages selected by the differentiating progeny of stem cells probably involve aspects of the transcriptional control of lineage-specific genes. Greaves and colleagues proposed that several lineage-specific genes are accessible to transcription factors or 'primed' in uncommitted cells. Accordingly, individual primitive cells were found to exhibit low levels of transcription of lineage-affiliated genes. Moreover, single stem cells expressed low levels of several of these genes, indicating that final lineage selection had not yet occurred. The multilineage 'priming' of stem cells is supported experimentally by the results of replating colonies composed of blast cells. This revealed that the blast cells themselves were bipotent or oligopot-ent progenitors for various lineages of blood cell development, and that the combinations of lineages found within individual colonies appeared to be randomly distributed, although some combinations are more common than others.

It is likely that differences in the expression levels of transcription factors determine the lineage affiliation of a differentiating cell (Figure 1.6). The transcription factors PU1 and GATA-1 have been implicated in myeloid and erythroid/megakaryocyte lineage specifications respectively. The common precursors of the myeloid, erythroid and megakaryocytic lineages coexpress PU1 and GATA-1, but GATA-1 is downregulated during myeloid cell development and PU1 during erythroid/megakaryocytic cell development. The decision of bipotent granulocyte/monocyte precursors to proceed along the granulocytic or monocyte macrophage lines of differentiation is influenced by C/EBP-a, which is required for granulocytic cell development.

Stem cell plasticity

Reports that transplanted bone marrow cells can contribute to the repair and regeneration of a spectrum of tissue types including brain, muscle, lung, liver, gut epithelium and skin have

Stem cell

PU.1 and GATA-1

Mixed myeloid progenitor (CFU-GEMM)

PU.1

CFU-GM

GATA-1

BFU-E/Mk

C/EBP-a

PU.1

GATA-1

i CFU-E

CFU-G

i CFU-E

CFU-Mk a

Neutrophils Monocytes/ macrophages

Eosinophils Platelets

Figure 1.6 Transcription factors involved in lineage selection by haemopoietic stem and progenitor cells.

attracted considerable attention. The important implication of these observations is that haemopoietic stem cells could be used clinically for tissue replacement therapies. The multipotential nature of haemopoietic stem cells appears to be well suited to a wider role in tissue repair as they already demonstrate the capacity to make renewal, differentiation and lineage choices. Moreover, cultured blood cells can transdifferentiate from one lineage to another. Cell fusion is an alternative mechanism accounting for the contribution of bone marrow cells to tissue repair (e.g. after myocardial infarction), possibly involving the macrophage component of the marrow infusion.

A second bone marrow stem cell population with tissue-regenerating potential, the multipotent adult progenitor cells (MAPCs), representing a subpopulation of mesenchymal (stromal) stem cells, was isolated by Verfaillie and colleagues. The MAPCs develop over a period of time in culture, during which they seem to lose tissue-restricted gene expression and become able to differentiate into mesenchymal cell types (osteoblasts, chondrocytes, adipocytes and sketetal myoblasts) endothelium, neuroectoderm and hepatocytes.

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