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A. Amoah et al.
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Figure 2. Kinetics of single cell division of young (blue) and aged (green) hematopoietic stem cells cultured ex vivo. (A) Experimental design of cells singly sorted into plates containing M4 media and incubated at 37°C, 3% oxygen. (B) Cumulative first division of live young and aged hematopoietic stem cells (HSC) and (C) time by which 50% of the cells have undergone the first division. *P=0.03; t-test with Welch’s correction. Bars represent the mean ± standard error the mean (SEM). (D) Cumulative second division of live young and aged HSC. (E) Slope was derived as linear regression fits and probability values calculated from the correlation coeffi- cients (M4 calculations in the box). Each curve was derived from cumulative gaussian fits with robust regression. nyoung=6; naged=5 different donors. Donor age: young =27-39 years (yr), median =28 yr; aged =64-75 yr, median =69 yr.
(Online Supplementary Figure S3A and B). The proportions of young and aged HSC in the G0, G1 and S-G2-M phases of the cell cycle were similar, hence the delayed initiation of division was not simply driven by a higher frequency of quiescent cells upon aging. In aggregation, aged HSC show a delayed initiation of division that is not linked to a higher frequency of cells in G0 or G1, while once division is initiated, there is no difference in the overall kinetics of the division.
Cdc42 activity is increased in human hematopoietic stem cells from the elderly and correlates with an increase in hematopoietic stem cell frequency
The small RhoGPTase Cdc42 cycles between an active (GTP bound) and inactive (GDP bound) state24 and has been shown to have essential roles in HSC regulation.25 We previously demonstrated that the active form (GTP- bound form) of the small RhoGTPase Cdc42 was increased in murine low-density bone marrow (LDBM) cells as well as in HSC upon aging, and that this increase in HSC resulted in the age-related increase in HSC fre- quency in mice.9 We therefore determined the level of activity of Cdc42 in human LDBM from the elderly by a
standard pull down assay (Figure 3A; Online Supplementary Figure S4A). Pull-down replicates on the same samples from randomized donors demonstrated overall high reproducibility of the pull-down assay also on primary human samples (Online Supplementary Figure S4B). We observed an approximate 4-fold increase in the relative Cdc42 activity in LDBM cells from elderly donors com- pared to the young (Figure 3B) which was re-affirmed in correlation analysis of Cdc42 activity and age (Online Supplementary Figure S4C). Using immunofluorescence imaging, we found aged HSC had significantly higher Cdc42-GTP expression than young HSC (Figure 3C to E; Online Supplementary Figure S4D), implying primitive stem cells also undergo Cdc42 activity changes upon aging. In addition, we identified a positive association between Cdc42 activity and HSC frequency but not between Cdc42 activity and HSPC frequency (Figure 3F and G). Furthermore, the spread of values and, thus, the standard deviation (Figure 3B) was higher in the aged group than in the young, which points to a strong increase in hetero- geneity of the hematopoietic system in individuals upon aging. Heterogeneity upon aging in mice is, for example, less pronounced due to their inbred nature.26–28 In aggrega-
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haematologica | 2022; 107(2)