170 ASSESSING THE POTENTIAL OF STEM CELLS TO GENERATE CHIMERIC RATS

Abstract

Embryonic stem (ES) cells are pluripotent cells derived from inner cell masses (ICMs) of blastocysts. The capacity of pluripotency in differentiation is assumed to contribute to embryonic development to form a chimeric individual when these cells are reintroduced into embryos. Chimeric mice can be routinely generated by aggregation of ES cells with morulae or injection into blastocysts, which are then implanted in pseudopregnant foster mothers. Furthermore, recent studies have demonstrated that bone marrow-derived stem cells and neural stem cells can integrate into the embryonic development in mouse (Geiger et al. 1998 Cell 93, 1055–1065; Clarke et al. 2000 Science 288, 1660–1663). We therefore tried to assess the ability of rat ICMs and neural stem cells to form chimeras by injecting these cells into rat blastocysts. Forty-two rat ICMs from Day 6 blastocysts of Dark Agouti (DA) inbred rat were injected into Day 5 blastocysts of Sprague-Dawleyd (SD) outbred rats; 14 pups were born following embryo transfer of these blastocysts injected into Hooded Wistar (HW) recipients. One male of the 14 pups was coat color-patched and displayed germline transmission. Following embryo transfer of 22 SD blastocysts injected by Day 5 DA ICMs, 7 pups were born and 2 of them were coat color-patched. Nine pups were obtained from 23 DA blastocysts injected by Day 5 SD ICMs; 4 of them were coat color-patched. The ICM cells were isolated and cultured for 6 days. No chimeras were generated by injection of the cultured ICM cells, as assessed by coat color patching. These results suggest that rat embryonic ICMs have potential to develop into chimeras, but the chimeric potential of ICMs was rapidly lost in our culture system. Investigation of potential chimeric development of rat fetal neural stem (rFNS) cells transfected with Lac Z was carried out. Staining was observed in tissues from 2 of 41 E14 fetuses. These results demonstrated that rFNS cells can integrate into the early embryonic environment although the ability of these cells to contribute to chimeric formation was marginal. No coat color chimerism was observed in any of the 88 pups generated from the LacZ-rFNS cell experiments.