(A) Summary of total human reconstitution in the bone marrow of NSG mice 16 weeks post transplantation of all the progeny cells from 50,000 CD34+ input cells that were transplanted freshly (Day 0) or co-cultured for up to 7 weeks. Shown are all individual recipients and mean value. P value was calculated for each time-point in comparison to Day 0. (** p<0.01). (B) Representative FACS plot of bone marrow and spleen from NSG recipient mice showing long-term multilineage engraftment of 50,000 input CD34+ FL cells that were isolated freshly or co-cultured 2 weeks on OP9M2. (C) Comparison of engraftment capacity between purified CD34+CD38−CD90+, CD34+CD38−CD90-, CD34+CD38+CD90+ and CD34− cells 2 weeks after co-culture demonstrates that the engraftable HSC are maintained only in the CD34+CD38−CD90+ fraction. Each mouse was transplanted with the respective population derived from 50,000 input CD34+ human fetal liver cells in culture. Shown are all individual recipients and mean value from one experiment (** p<0.01, * p<0.05). (D) Limited dilution assays (n = 2) was performed to estimate NSG-RC frequency among fetal liver CD34+ cells that were transplanted freshly or after 2 week culture. Mice were transplanted with equal number of input CD34+ cells at different concentrations and analyzed 16 weeks post transplantation. No significant difference in NSG-RC number (Day 0∶1 in 2610, upper 4821, lower 1413 and 2 wk: 1 in 3003, upper 5953, lower 1515) (p = 0.77) was detected.