Figure 3.

Il3 and Thpo have prosurvival and proproliferation effects and can expand HSCs. (A) The number of ckit⁺CD45⁺ AGM cells recovered after 3 days of explant culture in the presence or absence of Il3 or Thpo. (B) The percentage of live (7AAD⁻Annexin V⁻), early apoptotic (7AAD⁻Annexin V⁺), and dead (7AAD⁺Annexin V⁺) cells within the ckit⁺CD45⁺ population was determined. n = 3. (C) The percentage of proliferating cells that had incorporated BrdU during the last night of explant culture was determined within the ckit⁺CD45⁺ population. n = 3. (D) Repopulation levels of individual mice injected with AGM cells (0.1-0.3 embryo equivalents) explant-cultured in the presence or absence of Thpo. Dotted line represents 5% threshold. The number of positive mice out of total injected mice is indicated at the top. (E) Repopulation levels of individual mice injected with AGM cells explant-cultured in the presence or absence of Il3 and/or Thpo. Dotted line represents 5% threshold. The number of positive mice out of total injected mice is indicated at the top. (F) Multilineage analysis of donor cell contribution in one mouse highly repopulated with AGM cells cultured in the presence of Il3 and Thpo. (G) HSPCs (ckit⁺CD45⁺CD41^intermediate) were sorted from uncultured AGMs of the indicated genotypes and analyzed by qPCR for Id gene expression. n = 3. (H) AGMs were cultured in the presence or absence of 100 ng/mL Il3 or Thpo, and ckit⁺CD45⁺CD41^intermediate cells were sorted and then analyzed for the expression of Id1, Id2, or Id3 by quantitative real-time PCR analysis. n = 3. **P < .01, *P < .05.