The study of primitive hematopoietic cells and evaluation ot gene transfer protocols are facilitated by the use of assays that permit in vivo analysis (if transduced cells. Despite the many useful features of xenograft transplantation in fetal sheep, it remains unclear whether transduced human hematopoietic progenitors/stem celts can engratt <md proliferate in sheep/human chimeras. We transduced purified human CD341 bone marrow, or cord blood cells with a recombmant retroviru1(vMR1I-'AC) by coculture and transplanted the transduced cells via intraperitoneal injection into 5 sheep fetuses. In order to determine whether transduced human cells engraft in fetal sheep, three fetuses were sacrificed at da v 1.KM40 gestation, (80-S da\s after transplantation). "I wo of three fetuses analyzed contained both humanspecific β-globin and proviral sequences in the peripheral blood, bone marrow and spleen at autopsy indicating engraftment of transduced human hematopoietic cells- In order to evaluate the proliferation potential of the transduced cells, two lambs are being analysed sequentially. Bone marrow and peripheral blood samplings from one sheep have been obtained from 3 to 18 months after birth. Fluorescence activated cell sorting was performed to isolate human 'I cells (CD.V) ond C1W cells. In addition, human progenitors were cultured from bone marrow cells. Integration of the retrovirus in human cells was demonstrated by polymerase chain reaction ot proviral sequences in both human 1' cells and in individual progenitors. Inverse PC'R on individual progenitors was also performed to analyze the proliferation of individual primitive progenitor cells as a function of time. Three unique integration sites, indicating the proliferation of three distinct progenitor/stem cells were detected at times ranging from t> to at least 18 months of age. One integrant was detected in progenitors from 4 samplings over 10 months (b to 16 months) indicating long-lived proliferation of a primitive cell. These data demonstrate that transduced human hematopoietic progenitor cells are capable of engraftment in fetal sheep. I'he data also demonstrate that individual human hematopoietic progenitor/stem cells have the capacity to proliferate in vivo in this model for prolonged periods.
|Original language||English (US)|
|Number of pages||1|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology
- Cancer Research