Loss of ARHGEF2 function in CD34⁺ HSCs results in significantly diminished xenografts. (A) Immunofluorescent staining of ARHGEF2 and TUBA at the mitotic spindle in THP-1 and LAMA-84 cell lines. (B) Schematic of shRNA knockdown of ARHGEF2 in CD34⁺ HSCs in vivo and in vitro. (C) Protein level knockdown validation of shRNAs against ARHGEF2. (D) Proliferation of CD34⁺ HSCs in vitro over 7 days. (E) Colony output of ARHGEF2 knocked-down HSPCs. (F-G) Decreased engraftment (F) and output of CD15⁺ myeloid cells (G) derived from CD34⁺ HSCs with comparable gene transfer levels receiving shRNAs targeting ARHGEF2. There were 5 recipients each derived from 1 CB sample. (H) Hypothetical model summarizing the role of Arhgef2 (red dots) in regulating the orientation of the HSC mitotic spindle within the niche when establishing hematopoiesis, the loss of which may contribute to bone marrow failure at the stem cell level. Error bars represent SEM. *P < .05; **P < .01.