CEACAM1 expression associates with a primitive HSC phenotype. (A) CEACAM1 mRNA expression in UCB cells expanded with UM171 for 7 days and sorted based on EPCR expression from CD34⁺CD45RA^- cells (left) or sorted based on ITGA3 expression from CD34⁺CD45RA^-CD90⁺CD133⁺EPCR⁺ cells (right). RNA sequencing data are from Fares et al,¹²  and Tomellini et al,¹⁶  respectively. Each dot represents a biological replicate, and the median is denoted by the crossbar. TPM, transcripts per kilobase million. ****P = 3.14e-14 for EPCR⁺ vs EPCR^med/-, ****P = 2.79e-05 for ITGA3⁺ vs ITGA3^- based on Kallisto/Sleuth analysis.²³  (B) Proportion of CD34⁺CEACAM1⁺ cells in EPCR⁺, EPCR^med, and EPCR^- (top panel) as well as in ITGA3⁺ and ITGA3^- populations from UCB cells expanded with UM171 for 7 days. Percentages represent the relative size of each population in depicted gates. Data presented as mean ± SD for 6 independent biological replicates. (C) Proportion of CEACAM1⁺ (green) and CEACAM1^- (red) cells in the CD34⁺CD45RA^-CD90⁺EPCR⁺ subset as well as in ITGA3⁺ cells gated from this subset (follow reference gating) following expansion with UM171 or DMSO for 7 days. Data presented as mean ± SD for 4 independent biological replicates. (D) Sorting strategy for experiments presented in (E-G). CD34⁺ UCB cells were expanded for 7 days with UM171 before sorting. Data presented as mean ± SD for 10 independent biological replicates. (E) In vitro analysis of progeny of cells sorted based on CEACAM1 and ITGA3 expression following strategy presented in panel D and reintroduced in culture for 7 additional days. The bar graph represents the proportion of indicated cell populations in culture. Data are presented as mean ± SD for 3 biological replicates. (F) Estimated frequencies (red lines) presented as 1 per number of sorted cells and 95% confidence intervals (CIs) (gray boxes) of clonogenic cells in the indicated populations are shown. Cells were plated at 100, 10, and 1 cell per well. Cells were sorted from expanded cells from 2 separate UCB samples. *P = 0.05, (Mann-Whitney U test, 1-tailed). The objective of these experiments was to determine if the clonogenic cell frequency of the CEACAM1⁺ITGA3⁺ population is higher than that of the other subsets. The hypothesis assumed for this test is unidirectional, supporting the use of a 1-tailed test.