HSC aging and differentiation are associated with opposite expression programs. A joint PCA was performed for all noncycling cells, and each of the top two PCs distinguishes cells by their cell type and age, with higher scores for young and differentiated HSCs and lower scores for old and less-differentiated cells. (A–D) Each plot shows the loadings of PC1 and PC2, colored based on their cell type and age for cells from all six populations (A) or from specific pairs of populations that differ by age (B), differentiation (C), or both (D). (E) Distribution of PC1 + PC2 scores for young (top) and old (bottom) LT-HSCs and ST-HSCs. Aging is associated with a decrease in the PC1 + PC2 scores, and differentiation is associated with an increase in the PC1 + PC2 scores. (F) Colony formation assays using methylcellulose. Two hundred fifty of either young or old LT-HSCs and ST-HSCs were plated on methylcellulose (n = 5). Colonies were counted on day 10. The colony numbers are averages of duplicate measurements of each individual mouse. Statistically significant differences are as follows: (***) P < 0.001, (*) P < 0.05. (G) Distribution of the megakaryocyte progenitor (MkP) signature scores, defined as the average normalized expression of MkP-enriched genes (Sanjuan-Pla et al. 2013) (x-axis) for LT-HSC (red), ST-HSC (blue), and MPP (green) in young (top) and old (bottom) mice.
