Properties Of Hematopoietic Stem Cells

The hematopoietic stem cell (HSC) is the ultimate progenitor of all of the cells found in the peripheral blood. In mammalian systems, small numbers of HSC have been shown to be capable of extensive proliferation, generating millions of mature blood cells in regulated numbers each day. HSCs are multipotent and differentiate into cells of the erythroid (red cell), megakaryocytic (platelets), myeloid (granulocytes and mono-cytes), and lymphoid (B- and T-cell) lineages (Fig. 1). HSC can self-renew without differentiating, generating pluripotent progeny, which themselves can proliferate and differentiate into mature blood cells, [for reviews see (1-4)]. The ability of HSCs to self-renew allows the transplantation of a small number of HSCs to reconstitute the entire hematopoietic system of patients whose bone marrow had been ablated by chemotherapy or radiation (5-7).

The hematopoietic stem cell is especially attractive as a target for gene therapy of hematopoietic diseases (8-10). In theory, a small number of HSCs could be exposed to gene transfer vectors ex vivo and returned to a myeloablated recipient. Repopulation of the recipient with gene corrected HSC

would ensure a lifetime supply of modified peripheral blood cells of all lineages. If the transferred gene (transgene) were expressed at the appropriate levels in mature hematopoietic cells carrying the transgene, gene therapy could be the treatment of choice for many inherited and acquired hematopoietic diseases (8-10). In addition, recent evidence has suggested that the most primitive mouse bone marrow cells can give rise to cells of the vascular endothelium, and perhaps to many solid tissues as well (11-14). Thus the therapeutic potential of HSC may extend beyond the treatment of hematopoietic diseases. At this point, the experiments suggesting that primitive hematopoietic cells can directly transdifferentiate into functional cells other than the vasculature should be considered exciting but preliminary, as alternate explanations are possible (15).

As with any novel therapy, a careful examination of possible negative outcomes should also be considered. Many hema-topoietic malignancies arise as a result of mutations in primitive hematopoietic cells with great proliferative potential. In fact, many of the gene transfer vectors used to deliver new genetic material to HSC are derived from viruses first identified as pathological agents (16,17). The many modifications made to these viruses to negate possible pathological consequences is the subject of other chapters, but it should be remembered that even the most highly engineered vector still contains viral elements that can cause disease.


The definitive assay for mouse hematopoietic stem cells is the repopulation of stem cell deficient mice with transplanted hematopoietic cells (1,3,4). Repopulation by donor cells, as opposed to repopulation by residual host cells, is demonstrated

Figure 1 A model of hematopoiesis. Hematopoietic stem cells (HSC) can either self-renew or give rise to progenitor cells committed to either myeloid (CMP) or lymphoid (CLP) differentiation. As differentiation progresses, hematopoietic cells proliferate (not shown) and become progressively restricted to specific lineages of cells.

Figure 1 A model of hematopoiesis. Hematopoietic stem cells (HSC) can either self-renew or give rise to progenitor cells committed to either myeloid (CMP) or lymphoid (CLP) differentiation. As differentiation progresses, hematopoietic cells proliferate (not shown) and become progressively restricted to specific lineages of cells.

by analysis of genetic markers that differ between the donor and recipient mouse. The genetic markers used include chromosomal translocations (18), isozyme polymorphisms (19,20), Y-chromosome-specific DNA sequences (21), cell surface markers (22), and combinations of these. Multilineage repopulation of lethally irradiated hosts is demonstrated by the detection of the host genetic marker in cells of the different hematopoietic lineages (3,4).

The repopulation of recipient mice with limiting numbers of bone marrow cells has shown that HSC are a rare population of cells. For example, 1 study transplanted bone marrow cells from female mice heterozygous for the X-linked isozyme marker Phosphoglycerate Kinase (Pgk) into stem-cell-deficient W/Wv mice (19,23). Due to random inactivation of 1 X chromosome, individual HSC from heterozygous Pgk-a/b mice express either Pgk-a or Pgk-b. The peripheral blood cells of mice repopulated with large numbers of bone marrow cells contained an equal amount of Pgk-a and Pgk-b. In animals repopulated with successively fewer HSC, the relative contribution of individual HSC becomes greater. As predicted, the peripheral blood cells of individual mice repopulated with limiting numbers of bone marrow cells contained high levels of either Pgk-a or Pgk-b. This work and related studies demonstrated that a single HSC could repopulate a mouse, but that repopulation required an average of 1X 105 bone marrow cells (19,20,24). By contrast, limiting dilution assays have shown that the concentration of the repopulating stem cell in the bone marrow is more than 10-fold less than the concentration of the Colony Forming Unit-Spleen (CFU-S) (25), a multipotent cell that forms a mixed colony in the spleen of irradiated mice. The concentration of repopulating stem cells is also 10-fold less than the cell that is required for the 30-day survival of irradiated mice (3).

Competitive repopulation assays, in which mixtures of genetically distinguishable hematopoietic cells injected into stem-cell-deficient mice allow the relative ability of each population of cells to repopulate recipient animals can be quantified (26,27). These assays are a powerful tool for identifying, characterizing, and quantifying HSC.

B. Phenotype of Mouse Hematopoietic Stem Cells

The advent of Fluorescence Activated Cell Sorting (FACS) and the development of monoclonal antibodies that recognize specific markers expressed on the surface of hematopoietic cells have made it possible to separate the rare HSC from the large number of more mature hematopoietic cells. Spangrude et al. (22) demonstrated that mouse HSC do not express antigens present on surface of mature cells of the different hematopoietic lineages. Lineage marker negative (Lin -) cells represent less than 10% of bone marrow cells. Further enrichment of HSC was achieved by selecting Lin - cells expressing low levels of Thy-1.1 and the Sca-1 (Stem Cell Antigen) marker. Lin - Thy-1.1to Sca-1 + cells comprise less than 1 % of Lin -cells and are highly enriched for HSC and other primitive progenitor cells, including CFU-S and radioprotective cells (22).

The Sca-1 marker is expressed in about 50% of all inbred mouse strains, and the Thy-1.1 allele is present in only a few inbred strains (28). The Sca-1 and Thy-1.1 markers are found together in only 1 strain of mice (29). Several groups have shown that c-kit, the receptor for the hematopoietic growth factor SCF (Stem Cell Factor), can be used to discriminate between HSC and more mature hematopoietic cells (30-34). In most strains of mice, Lin - cells expressing high levels of c-kit, (c-kitHI) are highly enriched for HSC (30). In strains expressing Sca-1, the Lin- c-kitHI cells are also Sca-1 positive (33). HSC can also be distinguished from primitive progenitors that give rise to myeloid or lymphoid cells, the common myeloid progenitor (CMP) (35) and common lymphoid progenitor (CLP) (36) respectively. CMP are Lin- and c-kit + , but do not express Sca-1 or the Interleukin 7 receptor (IL-7R-). CLP are Lin-, c-kit + and IL-7R + . The most accurate and widely accepted phenotype for mouse HSC is Lin-, IL-7R-, c-kit+ , Sca-1 + (Fig. 1). An average of 30-50 cells enriched using these markers will repopulate 100% of recipient mice (3,4).

The fluorescent dyes Rhodamine 123 and Hoechst 33342 are pumped out of cells by ABC transporter proteins, primarily ABCG2, which is expressed at high levels in HSC (37-40). Many groups have used the Rhodamine 123 dye in combination with other markers to identify primitive hematopoietic cells, and Lin- cells that retain low levels of Hoechst 33342 (HoLO) and Rho 123 (RhoLO) are enriched for HSC (41-43). A novel method to enrich HSC analyzes the blue and green fluorescence of bone marrow cells stained with Hoechst 33342 only. A rare ''side population'' of cells with low levels of blue and green fluorescence has been shown to be highly enriched for HSC (44). Side population cells express c-kit and Sca-1, and are Lin- (44). Finally, using a fluorescent substrate for aldehyde dehydrogenase (ALD), Jones et al. demonstrated that a population of small, Lin -, hematopoietic cells that express high levels of ALD were highly enriched for HSC (45). Injection of an average of 10 small Lin- ALD positive cells led to the repopulation of 100% of recipient mice.

C. Sources of Mouse Hematopoietic Stem Cells

During mouse development, hematopoiesis begins in the yolk sac at day 8.5 of the 21-day gestation period (23,46,47). Yolk sac hematopoiesis generates only nucleated erythrocytes containing embryonic hemoglobin. Yolk sac hematopoietic cells are capable of repopulating chemically ablated newborn mice (48,49), but do not repopulate adult animals (50). The first HSC capable of repopulating lethally irradiated adult mice are found in the aorta-gonad-mesonephros (AGM) region of mouse embryos at day 11.5 of gestation (50). AGM region HSC are negative for lineage specific markers and express c-kit (50). From day 12.5 to day 17.5 of gestation, the fetal liver becomes the site of hematopoiesis, producing mature erythroid, myeloid, and lymphoid cells. Fetal liver HSC are lineage marker negative, express c-kit, and the marker AA4.1 (51). After day 17.5, the fetal spleen becomes the primary site of hematopoiesis (23), and by the time of birth, the bone marrow has become the primary site of hematopoiesis. In adult mice, HSC are found in the bone marrow, spleen, and peripheral blood (23,52-54). Competitive repopulation assays have been used to estimate that approximately 80% of the HSC are found in the bone marrow, 19% of the HSC reside in the spleen, and less than 0.5% of HSC are found in the peripheral blood (53,54).

The relative and absolute number of HSC in the peripheral blood of mice can be manipulated by treatment of animals with either hematopoietic growth factors or anti tumor agents such as cyclophosphamide (CP). The redistribution of HSC and progenitor cells into the peripheral blood is termed mobilization (55). For example, in mice treated with Granulocyte-Colony Stimulating Factor (G-CSF) for 7 days, approximately 10% of the HSC are found in the bone marrow, 88% of the HSC reside in the spleen, and 2% of HSC are found in the peripheral blood (53,56,57). Mice treated with FLT3 ligand (FL) and G-CSF for up to 10 days showed greater than 200fold increases in the repopulating ability of the peripheral blood compared to normal mice (58-60). Eight days after treatment with CP, the relative number of HSC in the peripheral blood is increased nearly 30-fold to a level similar to the HSC content of untreated bone marrow (61). Combinations of cytokines have even more pronounced effects on the level of peripheral blood stem cells, particularly in splenectomized mice. Treatment of splenectomized mice with G-CSF and SCF for 5 days causes a 3-fold increase in the total number of HSC, with 81% of the repopulating ability in the peripheral blood and 19% in the bone marrow (56,57). Similar results have been described with numerous other cytokines and cyto-toxic drugs (58-61). An unexpected finding in mice treated with G-CSF and SCF was a greater than 10-fold increase in the repopulating ability of the bone marrow 14 days after cytokine treatment was discontinued (62).

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