The engraftment of nonhuman primate hematopoietic cells was analyzed in five groups of irradiated NOD/SCID mice. As shown in Fig. 

6

, differentiated primate cell clusters were not detected by flow cytometry in the PB and BM cells of negative control mice (saline) in week 8 post transplantation. On the other hand, the primate CD45 marker was detected at 4.92 ± 1.24 %, 1.89 ± 0.43 %, 1.47 ± 0.62 %, and 7.84 ± 2.31 % in the PB of groups transplanted with day 0 primate CD34

+

cells, day 9 expanded primate CD34

+

cells, day 9 GFP-control lentiviral transduced CD34

+

cells, and day 9 GFP-Sall4B lentiviral transduced CD34

+

cells, respectively (Fig. 

6a

). Primate myeloid lineage marker CD14 and lymphoid lineage marker CD20 could also be detected in the cell transplanted groups (Fig. 

6a

). In mouse BM, primate CD45 marker was exhibited at 5.27 ± 1.04 %, 2.73 ± 0.77 %, 2.45 ± 0.92 %, and 7.01 ± 1.18 % in day 0 primate CD34

+

cells, day 9 expanded primate CD34

+

cells, day 9 GFP-control lentiviral transduced CD34

+

cells, and day 9 GFP-Sall4B lentiviral transduced CD34

+

cells, respectively (Fig. 

6b

). Primate lineage markers CD14 and CD20 were also detected at different levels in four cell transplanted groups (Fig. 

6b

). These results indicated that GFP-Sall4B transduced cells had the ability to control their differentiation, repopulation, and peripheral cell output, when compared with GFP-control transduced cells or normal expanded CD34

+

cells on day 9 in vivo

.

The cell population expanded by Sall4B ex vivo for 9 days was revealed to have priority in repopulating primate cells in NOD/SCID mice. Moreover, the SRC frequency was found to be significantly enhanced by stem cell factor Sall4B through limiting dilution analysis, which demonstrated a 2.27-fold increased SRC frequency in mice that received 9-day-old SALL4B expressed primate CD34

+

cells (59 per 10

6

starting CD34

+

cells) compared with those receiving 9-day-old lentivirus control primate CD34

+

cells (26 per 10

6

starting CD34

+

cells), and a 1.59-fold increased compared with those unexpanded primate CD34

+

cells (37 per 10

6

CD34

+

starting cells) at 8 weeks post transplantation. These results suggested that the Sall4B could be effective in expanding nonhuman primate CD34

+

cells.

Fig. 6

Nonhuman primate cells in PB and BM of NOD/SCID mice transplanted with various stages of CD34+ cells. Eight weeks after intravenous primate CD34+ transplantation in mice, primate CD45+, CD14+, and CD20+ cells were analyzed in the PB (a) and BM (b) of mice transplanted with unexpanded primate CD34+ cells, 9-day expanded CD34+ cells, 9-day GFP-control transduced CD34+ cells, and 9-day Sall4B-GFP transduced CD34+ cells, respectively. Normal saline was injected as the vehicle control. Data represent mean ± SD, n = 16. **P < 0.01. One-way ANOVA followed by Dunnett’s multiple comparison test. BM bone marrow, GFP green fluorescent protein, PB peripheral blood

To further determine whether Sall4B-transduced cells bear long-term engraftment, secondary BM transplantations were performed. Flow cytometry analysis of mouse BM cells harvested from femurs in week 8 post secondary transplantation showed that primate CD45

+

cells could be detected at 1.87 ± 0.73 %, 0.53 ± 0.22 %, 0.21 ± 0.12 %, and 2.05 ± 1.03 % in groups transplanted with day 0 primate CD34

+

cells, day 9 expanded primate CD34

+

cells, day 9 GFP-control lentiviral transduced CD34

+

cells, and day 9 GFP-Sall4B lentiviral transduced CD34

+

cells, respectively (Fig. 

7

). The result demonstrated that Sall4B-transduced CD34

+

cells could be successfully transplanted from one animal BM to another on day 9. It was verified that Sall4B-transduced CD34

+

cells possessed long-term engraftable property.

Fig. 7

Secondary BM transplantation in NOD/SCID mice. Representative flow cytometric analysis of the mouse BM CD45+ cell population from five groups in week 8 post secondary transplantation. NS normal saline, GFP green fluorescent protein