M1-MΦs and M2-MΦs differentially regulate HSC self-renewal and in vitro expansion via NOS2 and Arg-1, respectively. (A) Nos2 knockout abrogates the inhibitory effect of M1-MΦs on 5-week CAFCs. LSK cells (2 × 10³) were cocultured with 1 × 10⁵ M1-MΦs from wild-type mice and Nos2 knockout (Nos2^−/−) mice in StemSpan medium supplemented with 20 ng/mL SCF and TPO for 6 days. Numbers of total cells, LSK cells, and 5-week CAFCs in the input LSK cells and the progeny from these cultures are presented as mean ± SEM (n = 3 independent cultures). ^aP < .05 vs Input, ^bP < .05 vs M1-MΦ, unpaired Student t test. (B) Arg1 knockout abrogates the promoting effect of M2-MΦs on 5-week CAFCs. LSK cells were cocultured with M2-MΦs from wild-type mice and Arg1-knockout (Arg1^−/−) mice, as described in panel A. The data are mean ± SEM (n = 3 independent cultures). ^aP < .05 vs Input, ^bP < .05 vs Input and M2-MΦ, unpaired Student t test. (C) Spermidine dose dependently increases the expansion of LSK cells and 5-week CAFCs in vitro. LSK cells were cultured with increasing concentrations of spermidine, as described in panel A. Data are mean ± SEM (n = 3 independent cultures). ^aP < .05 vs Input, ^bP < .05 vs 0 µM, ^cP < .05 vs 1 µM, ^dP < .05 vs 5 µM, 1-way ANOVA. (D) Relative gene expression in LSK cells sorted from the progeny of LSK cells cultured with M1-MΦs and M2-MΦs compared with that of input LSK cells revealed that coculture with M2-MΦs upregulated the expression of several HSC self-renewal and antiapoptotic genes, whereas coculture with M1-MΦs had opposite effects and increased the expression of the proapoptotic protein Bax. Data are mean ± SEM (n = 3 independent cultures). *P < .05, ***P < .001 vs cells cultured with M1-MΦs, unpaired Student t test. (E) Representative flow cytometric analysis of apoptosis (left panel) and percentage of apoptotic cells (right panel) in input LSK cells and LSK cells after culture with M1-MΦs or M2-MΦs or without MΦs. Data are mean ± SEM (n = 2 independent cultures). ***P < .001 between the cells cultured with M1-MΦs and all other cells, unpaired Student t test. (F) Hypothetical model illustrating the role of MΦ polarization in the regulation of mouse BM HSC self-renewal and expansion in vitro.