Aging of human hematopoietic stem cells is linked to changes in Cdc42 activity

Abstract

In this study, we characterize age-related phenotypes of human hematopoietic stem cells (HSC). We report increased frequencies of HSC, hematopoietic progenitor cells and lineage negative cells in the elderly but a decreased frequency of multi-lymphoid progenitors. Aged human HSC further exhibited a delay in initiating division ex vivo though without changes in their division kinetics. The activity of the small RhoGTPase Cdc42 was elevated in aged human hematopoietic cells and we identified a positive correlation between Cdc42 activity and the frequency of HSC upon aging. The frequency of human HSC polar for polarity proteins was, similar to the mouse, decreased upon aging, while inhibition of Cdc42 activity via the specific pharmacological inhibitor of Cdc42 activity, CASIN, resulted in re-polarization of aged human HSC with respect to Cdc42. Elevated activity of Cdc42 in aged HSC thus contributed to age-related changes in HSC. Xenotransplant, using NBSGW mice as recipients, showed elevated chimerism in recipients of aged compared to young HSC. Aged HSC treated with CASIN ex vivo displayed an engraftment profile similar to recipients of young HSC. Taken together, our work reveals strong evidence for a role of elevated Cdc42 activity in driving aging of human HSC, and similar to mice, this presents a likely possibility for attenuation of aging in human HSC.

Figure 1.

Flow cytometric analysis of different bone marrow populations isolated from young (white) and aged (grey) donors. Populations of interest within the lowdensity mononuclear cell fraction (MNC) were identified and frequencies of (A) Lin-CD34+^{ssc low}, (B) hematopoietic stem progenitor cells (HSPC), (C) hematopoietic stem cells (HSC) and (D) Lin- cells from young and aged donors were determined. *P<0.04, **P<0.005; Mann-Whitney and t-test with Welch’s correction. Bars represent the mean ± standard error of the mean (SEM). (E) Representative image of common myeloid progenitor/ megakaryocyte–erythroid progenitor (CMP/MEP), multipotent progenitor (MPP) and multipotent lymphoid progenitor (MLP) gates. (F) The frequency of CMP/MEP, MPP and MLP in the Lin- fraction of donors. **P=0.003; Mann-Whitney test. Bars represent the mean ± SEM. 17< nyoung >20; 23< naged >29. Donor age: young =23-39 years (yr), median =27 yr; aged =58-82 yr, median =65 yr. nyoung: number of young donors; naged: number of aged donors.

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 coefficients (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.

Figure 3.

Relative expression of Cdc42 activity in young and aged hematopoietic stem cells. (A) Representative image of western blot. (B) Quantitative expression of relative Cdc42 activity normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). *P= 0.010, if the two points with activity higher than 20 are excluded, then P=0.017; Mann-Whitney test. Bars represent the mean ± standard error of the mean (SEM). nyoung=13; naged=41. Donor age: young =23-39 years (yr), median = 27 yr; aged = 60-82 yr, median = 66 yr. (C) Representative confocal image of Cdc42-GTP expression in hematopoietic stem cells (HSC). Cdc42-GTP quantification of young and aged HSC normalized to (D) DAPI intensity and (E) cell size. ***P=0.0001, **P=0.0012; Mann-Whitney test. Bars = mean ± SEM and nyoung=66; naged=67, from three different donors per cohort. Donor age: young =27-31 years (yr), median =27 yr; aged =63-76 yr, median =76 yr. Scale bar represents 2 mm. (F) Correlation analysis (Spearman) of relative Cdc42 activity and HSC frequency (r=0.4, P= 0.05, n=37) and (G) hematopoietic stem progenitor cell (HSPC) frequency (r= 0.3, P=0.175, n=22), broken grey lines represent 95% Confidence Interval.

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.

Figure 5.

Analysis of xenotransplantation experiment under non-irradiated settings. Frequency of human cells in the bone marrow (BM) of NBSGW mice at (A) 8 weeks and (B) 12 weeks post-transplant. (C) Emergence of human myeloid cells and (D) B cells post-transplant from young (blue), aged (green) and CASIN-treated aged (orange, brown) -recipients. Red line represents averages of non-transplanted controls. Bars represent the mean "±" standard deviation (SD). n = 6 different donors per cohort, 46 mice at 8 weeks and 42 mice at 12 weeks. Donor age: young =24-35 years (yr), median =27 yr; aged =60-81 yr, median =62 yr.