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The Human Sheep Xenograft Model for the Study of the In Vivo Potential of Human HSC and In Utero Gene Transfer

(summarized by Zhigang Gao and Anita Wokhlu)

A modification of the human sheep xenograft model has been developed for the in vivo evaluation of the engraftment and differentiation potential of human hematopoietic stem/progenitor cells. This exhaustion strategy involves the administration of human growth factors to hematopoietic chimeras which have been generated by transplantation of human hematopoietic cells into pre-immune fetal sheep recipients. Initial results indicate that long-term engrafting human stem cells (HSC) can be distinguished from committed progenitor cells by use of this strategy. The model can also be used to assess expansion and transduction of HSC.

The "late-acting" human specific growth factors, GM-CSF and interleukin 3, were administered to human-sheep hematopoietic chimeras on nine days of every month. Administration of these human cytokines was found to augment the levels of human cells in the bone marrow of the chimeras from about 5% to approximately 15%. Hypothetically, these cytokines elevate the levels of human cells in the chimeras by stimulating committed human progenitor cells, potentially "exhausting" these committed progenitor cells. In contrast, HSC should not be affected by these cytokines. If this is true, persistence of human cells of multiple lineages in chimeras despite this cytokine treatment may be able to be used to assess human HSC.

A comparison of the behavior of transplanted human CD34⁺CD38⁺ cell preparations (enriched in committed progenitor cells, depleted of HSC) versus CD34⁺CD38^– cell preparations (enriched in HSC) tests the utility of this system and hypothesis. In prior published experiments, human CD34⁺CD38^– cell preparations engrafted after transplantation to primary fetal sheep. Moreover, after the human cells from marrows of the primary sheep were transplanted into secondary fetal sheep hosts, human cells were present persistently in the secondary chimeras. In contrast, CD34⁺CD38⁺ cell preparations engrafted in primary recipients, but not upon secondary transfer. In the exhaustion experiments, chimeras which had been generated by transplantation of CD34⁺CD38⁺ cell preparations were quickly exhausted of their content of human cells. In contrast, chimeras generated by transplantation of CD34⁺CD38^– cell preparations exhibited delayed exhaustion. Dr. Zanjani reported that the exhaustion strategy can be used to assess the engraftment capability of a population of cells within four to six months. This is much faster than protocols currently used with the human-fetal sheep model to distinguish early human progenitor cells from HSC, which rely on long-term results of serial transplantation experiments.

The human-sheep xenotransplantation model was also used to assess the in vivo potential of CD34⁺ versus CD34^– human bone marrow cells. Although both CD34⁺ and CD34^– human bone marrow cells generated persistent engraftment of multiple lineages of human cells in the chimeras, chimeras generated by transplantation of 4 x 10⁴ CD34⁺ cells were "exhausted" by treatment with four cycles of human growth factors. In contrast, there was not significant "exhaustion" noted in chimeras generated by transplantation of 6 x 10⁴ CD34^– cells.

Using the exhaustion strategy, the persistence of HSC activity was found to depend on the source of cells, their phenotype, and their concentrations. As illustrated with the CD34⁺ and CD34^– populations, the human sheep xenograft model can be used to further characterize ex vivo expansion, homing, and transduction.

The permissive environment of the developing pre-immune fetal sheep allowed long-term transfer and expression of Neo^-R gene following the direct trans-maternal uterine injection of retroviral vectors into the fetus (i.p.). In utero transplantation of 6 x 10⁵ human CD34⁺ cells followed then by i.p. injection of a high titer retroviral vector resulted in gene transduction of human cells in both primary and secondary chimeric sheep. Of the human cell colonies, 8%-22% expressed Neo^-R gene by polymerase chain reaction detection. The levels of transduced cells were comparable in the primary and secondary recipients (up to 11 months after transplantation).

Thus, the human-sheep xenograft model provides a biologically relevant assay to determine the in vivo potential of stem cells and may also serve as a pre-clinical model for in utero gene therapy.

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