In Vivo Models

The ability of HSC to maintain lifelong hematopoiesis can only be assayed in vivo. Several animal models have been developed for assaying both murine and human HSC activity in vivo.

W/W mice have a spontaneous mutation in their c-kit receptor and die shortly after birth due to anemia and cytopenia unless rescued by transplantation with normal WBM (90). Thus, the systemic deleterious effects of lethal irradiation are avoided by using these mice as an in vivo model. W/W mice have been used to show that fetal liver contains HSCs capable of repopulating adult WBM (91).

Figure 3 Representative in vitro colonies. (A) Typical appearance of a WBM-derived myeloid colony cultured for 7 days in methylcellu-lose supplemented with fetal calf serum and cytokines. (B) CFU types can be characterized by Wright-Geimsa stain, which allow the visualization of cytoplasmic and nuclear morphology. In this representative experiment, a mixed CFU comprising macrophages (white arrow), differentiated granulocytes (black arrow), and progenitors (striped arrows) is visualized.

Figure 3 Representative in vitro colonies. (A) Typical appearance of a WBM-derived myeloid colony cultured for 7 days in methylcellu-lose supplemented with fetal calf serum and cytokines. (B) CFU types can be characterized by Wright-Geimsa stain, which allow the visualization of cytoplasmic and nuclear morphology. In this representative experiment, a mixed CFU comprising macrophages (white arrow), differentiated granulocytes (black arrow), and progenitors (striped arrows) is visualized.

To quantitatively evaluate in vivo HSC activity, donor and recipient PB reconstitution must be distinguishable because lethal irradiation does not ablate 100% of endogenous WBM. The CD45 system of congenic mice can simultaneously distinguish between donor and recipient and verify the PB identity of analyzed cells (92). CD45 is a cell surface tyrosine phos-phatase exclusively expressed by nucleated hematopoietic cells (93). Monoclonal antibodies can discriminate between multiple alleles of CD45. Typically, CD45.1 WBM cells are transplanted into irradiated CD45.2 mice, and flow cytometry is used to examine recipient PB for CD45.1 positive cells. Repopulation of the different compartments of the PB can be analyzed by costaining with PB lineage markers. This model can be made semiquantitative by injecting known numbers of both a ''test'' population of CD45.1-derived cells with ''competitor'' CD45.2-derived WBM into CD45.2 recipients. The HSC activity of the test population and the competitor WBM is then compared (94).

Figure 4 Summary of in vivo and in vitro primitive hematopoietic activity assays. Depicts which primitive hematopoietic stem cell and progenitor populations are assayed by various in vitro and in vivo assays.

Murine models have also been developed to examine the primitive hematopoietic potential of human WBM. In the SCID-hu model, pieces of either human fetal thymus or human fetal lymph nodes are implanted under the kidney capsule of SCID mice (95). SCID mice do not produce graft-rejecting lymphocytes and are therefore amenable to transplantation with non-self-tissues or cells. This model allowed the in vivo differentiation of human B and T cells to occur transiently (95).

Bg/nu/xid mice also do not produce graft-rejecting lymphocytes or natural killer (NK) cells (96). Transplantation of human WBM cells into sublethally irradiated bg/nu/xid mice yielded human lymphoid engraftment for 5 weeks. SCID mice injected with human growth factors and nonobese diabetes (NOD)/SCID mice (SCID mice crossed with nonobese diabetic mice that lack NK cells) can maintain higher and sustained levels of engraftment with human lymphoid cells (97,98). Also, mice transplanted with SCID WBM can support grafts of human lymphoid cells (99). Most human in vitro assays only detect myeloid differentiation because the architecture of the bone marrow and thymus, which are difficult to recreate in vitro, are required for B and T cell development. Thus, murine models of human hematopoietic differentiation are key to studying human lymphoid development in the laboratory.

Fetal sheep have also been used to assess human HSC activity in vivo. The immature immune system of the fetal sheep is tolerant to human cells transplanted in utero. Human fetal liver-derived HSC have been found to stably engraft the bone marrow and PB of primary and secondary fetal sheep recipients for >2 years posttransplant (100,101).

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