Embryonic Stem Cell Incorporation in Gastric Wall is Enhanced by Modified Proinflammatory Stem Cells and Enhanced Proliferative Host Compartment

BACKGROUND We showed previously that modified embryonic stem cells (ESCs) can be transplanted into and constitute mouse gastric wall for local delivery of particular gene products. We wished to assess differences in transplant efficiency depending on host and stem cell modifications. As a model, we looked at wild type (WT) and ITF-deficient (ITF) mice, since the latter are known to have an expanded proliferative compartment and are susceptible to epithelial injury factors presumed preferential for ESC incorporation. ESCs were compared to modified ESCs over-expressing inducible nitric oxide synthase (NOSCs). METHODS ESCs were derived from blastocysts of male B6;129S-Gt(ROSA)26Sor mice constitutively expressing β-gal activity. ESCs and NOSCs (5*106) were injected into the gastric wall of 10-12 week old female B6129F1 (Jackson Lab, WT) and ITF-/mice. Stomachs were harvested 7 or 10 days after transplantation. BrdU (50μg per gram of body weight) was injected i.p. in mice 2 hours before sacrificing. Tissues were fixed in 10% buffered formalin. ESCs and NOSCs were identified by X-gal staining and confirmed as male genotype by Y chromosome FISH (Starfish). Frozen or paraffin sections were stained with Sheep antiBrdU antibody conjugated with biotin (Abcam) and detected by DAB substrate kit (Vector). RESULTS Scattered clusters of ESCs and NOSCs were observed in gastric mucosa of both ITF-/and WT mice, but some injected cells were found densely at the base of gastric mucosa after 7 days. There was greater presence of NOSCs in ITF-/compared to WT mice. X-gal positive cells were situated higher along the crypt axis at day 10 compared to day 7. In contrast, ESCs showed similar pattern of residence in both ITF-/mice and WT mice. In WT mice, NOSCs and ESCs had similar density and distribution in the gastric wall 7 days after transplantation. However, in ITF-/mice, there was greater presence of NOSCs than ESCs at 7 days. Transplanted stem cells were confirmed by Y chromosome FISH. Proliferative capacity of tissues transplanted with stem cells was evaluated by BrdU uptake. In WT mice, there is no significant difference in number or distribution of BrdU-uptaking cells between ES and NOSC injected stomachs. However, in ITF-/mice, there was greater number of BrdU-uptaking cells when injected with NOSCs than ESCs, similar to their greater presence by X-gal stains. Overall, ITF-/mice had greater number of BrdU-uptaking cells than WT mice. CONCLUSION Both host and ES factors appear to influence the distribution and possible incorporation of injected cells. ITF-/mice, with an expanded proliferative compartment, may be capable of greater incorporation of ESCs. iNOS delivery in these mice, co-acting with their decreased toleration of inflammation, may explain the enhanced transplant efficiency.