Fig. 6: Functional characterization of CD273/PD-L2 expressing HSPCs.

a CD273 surface expression level on different HSPC populations (n = 7 donors) by flow cytometry. One-way ANOVA with Tukey’s multiple comparisons test. b–f CD273^high and CD273^low CD34⁺CD38⁻CD45RA⁻ HSPCs were FACS-sorted for analyses (see Fig. 5). b CD273^high HSPCs show delayed in vitro differentiation. Cells were analyzed after 7 days of culture by flow cytometry (n = 4 donors). Two-way ANOVA with Šídák’s multiple comparisons test c) Protein expression of CD273 and stem cell-related marker proteins in FACS-sorted CD273^high and CD273^low HSPCs determined by Simple Western technology. d Expression of stem cell signature genes measured by bulk RNA-Seq of FACS-sorted CD273^high and CD273^low HSPCs (n = 4 donors). Box extends from 25th–75th percentiles, marking the mean with a distinct line. The whiskers reach out to the maximum and minimum data point. Two-tailed Mann-Whitney test (THY1, BEX1, KLF4, DLK1) and two-tailed unpaired t-test (HOPX, MLLT3, MPL, CDK6). e Expression of stem cell signature genes measured on hierarchically gated CD273^high and CD273^low HSPCs from AbSeq analysis showed the same pattern as bulk RNA-Seq in d. f Videomicroscopy-based single cell tracking of FACS-sorted CD273^high and CD273^low HSPCs showed delayed entry into first cell cycle of CD273^high HSPCs (n > 35 cells). Two-tailed unpaired t-test. g Fold expansion of FACS-sorted CD273^high and CD273^low HSPCs after 3 days in vitro culture (n = 4 donors). Two-tailed unpaired t-test. h Colonies determined by colony-forming unit assay (CFU) after 14 days (n = 5 donors). Two-way ANOVA with Tukey’s multiple comparisons test. i Replating of primary CFU (n = 4 donors). Two-way ANOVA with Tukey’s multiple comparisons test. Bars represent the mean with SEM.