Figure 2.
CLPB-deficient HSPCs have impaired granulocytic differentiation. Human cord blood CD34+ cells were nucleofected with guide RNA targeting CLPB (CLPB-g1 or CLPB-g3) or a control guide RNA targeting the intron of AAVS1 (as a gene editing control) complexed together with recombinant Cas9 protein. Edited CD34+ cells were cultured for 7-14 days in media containing G-CSF and SCF. (A) Representative flow plots showing gating strategy to identify CD11b+CD16+ mature neutrophils (PMN) or CD11b+/−CD16− granulocytic precursors; data are gated on CD14− cells to remove monocytes. Data are quantified in the right panel. (B) Representative hematoxylin/eosin-stained cytospin preparations of cells on day 14 of culture; original magnification using a 63X objective. Data are quantified in the right panel. (C) Number of CFU-G per 2000 gene-edited CD34+ cells. (D) Fold change (from day 0) in edited cells (for CLPB, edited out-of-frame) or unedited cells (for CLPB, unedited plus edited in-frame) in PMNs or granulocytic precursors sorted on day 14. (E) Percentage of caspase-3+ granulocytic precursors on day 7 of culture. (F) Cells were cultured for 7 days, and cell cycle was assessed by flow cytometry. Data represent 3-5 independent experiments. *P < .05, **P < .01, ***P < .005, repeated measures 1-way ANOVA.

CLPB-deficient HSPCs have impaired granulocytic differentiation. Human cord blood CD34+ cells were nucleofected with guide RNA targeting CLPB (CLPB-g1 or CLPB-g3) or a control guide RNA targeting the intron of AAVS1 (as a gene editing control) complexed together with recombinant Cas9 protein. Edited CD34+ cells were cultured for 7-14 days in media containing G-CSF and SCF. (A) Representative flow plots showing gating strategy to identify CD11b+CD16+ mature neutrophils (PMN) or CD11b+/−CD16 granulocytic precursors; data are gated on CD14 cells to remove monocytes. Data are quantified in the right panel. (B) Representative hematoxylin/eosin-stained cytospin preparations of cells on day 14 of culture; original magnification using a 63X objective. Data are quantified in the right panel. (C) Number of CFU-G per 2000 gene-edited CD34+ cells. (D) Fold change (from day 0) in edited cells (for CLPB, edited out-of-frame) or unedited cells (for CLPB, unedited plus edited in-frame) in PMNs or granulocytic precursors sorted on day 14. (E) Percentage of caspase-3+ granulocytic precursors on day 7 of culture. (F) Cells were cultured for 7 days, and cell cycle was assessed by flow cytometry. Data represent 3-5 independent experiments. *P < .05, **P < .01, ***P < .005, repeated measures 1-way ANOVA.

Close Modal

or Create an Account

Close Modal
Close Modal