Figure 5.

FACS Purification and Quality Control of CD90⁺ HSPCs

(A) Schematic of the experimental design. (B) Flow cytometric assessment of cells before FACS (CD34⁺, first plot) and purified CD90⁺ cells after sorting on the Sony (second plot) and Tyto (plot). (C) Comparison of the purity, yield, and fold enrichment of CD90⁺ HSPCs on the Sony and Tyto sorters. (D) CFC potential of CD90⁺ cells within bulk CD34⁺ HSPCs and FACS-purified CD90⁺ subsets. CD90⁺ cells from all three conditions were sorted into CFC assays to exclude contaminating CD90^– cells. (E) Flow cytometric quantification of GFP-expressing CD90⁺ cells within bulk CD34⁺ cell and FACS-purified CD90⁺ subsets (left) and the delta-MFI of GFP expression in gene-modified cells (right). (F) Erythroid, myeloid, and erythro-myeloid CFC potential of gene-modified CD90⁺ cells from bulk CD34⁺ cells and FACS-purified CD90⁺ subsets. CD90⁺ cells from all three conditions were sorted into CFC assays. (G and H) Individual colonies from all three conditions in (F) were picked, and (G) the gene-modification efficiency in CFCs was determined by PCR as well as (H) the VCN in modified CFCs quantified by qPCR. (See also Table S9.) Statistics, means ± SEM; in C, third graph, median and range; significance values, two-tailed paired t test.