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A. Amoah et al.
of Cdc42 activity and Cdc42 polarity were performed on human HSC. The data revealed that indeed Cdc42 activity and the frequency of polar HSC were inversely correlated (Figure 4E), suggesting a causative link between Cdc42 activity and polarity also for human HSC.
Aged hematopoietic stem cells are repolarized by pharmacological inhibition of Cdc42 activity
CASIN (Cdc42 activity specific inhibitor) is a pharma- cological compound that specifically inhibits the activity of Cdc42.30,31 We thus tested whether inhibition of Cdc42 activity in aged human HSC via CASIN might result in an increase in the frequency of polar HSC. CASIN indeed increased the frequency of HSC polar for Cdc42 in a dose-dependent fashion (Figure 4F) but not for tubulin (Online Supplementary Figure S5C). Inhibition of Cdc42 activity, thus, increases the frequency of aged HSC polar
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for Cdc42, though not fully to the frequency seen in young HSC (Figure 4C). This data confirms that elevated activity of Cdc42 is, at least in part, causative for the reduced polarity of aged human HSC similar to aged murine HSC.8,9,32
CASIN treatment induces early division onset in aged hematopoietic stem cells
We previously demonstrated that HSC from aged donors were delayed in division onset in comparison to HSC isolated from the young (Figure 2B and C), hence we assessed the effect CASIN treatment would have on the division of aged HSC. Our results show that upon CASIN treatment, aged HSC commence division hours before untreated HSC (Figure 4G) with no additional effect on the second division (Online Supplementary Figure S6D to E). We observed that the time taken for 50% of untreated
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Figure 4. Hematopoietic stem cells polarity assessment and division kinetics in the presence and absence of CASIN. (A) Illustration of a polar and apolar cell. (B) Representative immunofluorescent images taken with a confocal microscope. Quantification of the proportion of young (white) and aged (grey) donor cells polar for (C) Cdc42 and (D) tubulin. ***P=0.00018, **P=0.002; t-test, false discovery rate approach. Bars represent the mean ± standard error of the mean (SEM). Scale bar represents 2 mm; nyoung>5; naged>8. Donor age: young =26-39 years (yr), median =28 yr; aged =58-82 yr, median =65 yr. (E) Correlation analysis (Pearson) of Cdc42 polarity and relative Cdc42 activity (r=0.6, P=0.03, n =13), broken grey lines represent 95% Confidence Interval. (F) Quantification of Cdc42 polarity of hematopoietic stem cells (HSC) after treatment with CASIN. *P=0.03; Mann-Whitney test. Bars represen the mean ± SD. naged>2. Donor age: aged =61-81 years (yr), median = 62 yr. (G) Cumulative first division of aged HSC with and without CASIN and (H) time by which 50% of the cells have undergone the first division. Curve was derived from cumulative gaussian fits with robust regression. naged and naged+CASIN=4. Donor age: aged =63-71 yr, median =66 yr.
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haematologica | 2022; 107(2)