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Aging alters the cell cycle control and mitogenic signaling responses of human hematopoietic stem cells

Hammond, Colin A., Wu, Si Wei, Wang, Fangwu, MacAldaz, Margarita E., Eaves, Connie J.·2023·Blood
DOI 10.1182/blood.2022017174
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Abstract

Human hematopoietic stem cells (HSCs), like their counterparts in mice, comprise a functionally and molecularly heterogeneous population of cells throughout life that collectively maintain required outputs of mature blood cells under homeostatic conditions. In both species, an early developmental change in the HSC population involves a postnatal switch from a state in which most of these cells exist in a rapidly cycling state and maintain a high self-renewal potential to a state in which the majority of cells are in a quiescent state with an overall reduced self-renewal potential. However, despite the well-established growth factor dependence of HSC proliferation, whether and how this mechanism of HSC regulation might be affected by aging has remained poorly understood. To address this knowledge gap, we isolated highly HSC-enriched CD34+CD38−CD45RA−CD90+CD49f+ (CD49f+) cells from cord blood, adult bone marrow, and mobilized peripheral blood samples obtained from normal humans spanning 7 decades of age and then measured their functional and molecular responses to growth factor stimulation in vitro and their regenerative activity in vivo in mice that had undergone transplantation. Initial experiments revealed that advancing donor age was accompanied by a significant and progressively delayed proliferative response but not the altered mature cell outputs seen in normal older individuals. Importantly, subsequent dose-response analyses revealed an age-associated reduction in the growth factor–stimulated proliferation of CD49f+ cells mediated by reduced activation of AKT and altered cell cycle entry and progression. These findings identify a new intrinsic, pervasive, and progressive aging-related alteration in the biological and signaling mechanisms required to drive the proliferation of very primitive, normal human hematopoietic cells.

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Figure 1
Figure 1 CC-BY-NC-ND · From the PDF
Figure 2 Similar in vivo repopulating activity of neonatal and adult CD49f + cells when cotransplanted with CD34 + CB ' helper ' cells. (A) Design of a cotransplantation strategy to compare the regenerative potential of CD49f + cells isolated from donors of different ages. (B) Flow-cytometric analysis of the cells in a representative BM aspirate obtained from a mouse that had undergone cotransplantation 12 weeks earlier with CB CD49f + cells and GFP-labeled CD34 + CB cells. (C-D) Percentage of human CD49f + cell (GFP -) (C), and NM, B-lymphoid, erythroid, and CD34 + progeny (D) from input CB, young adult BM, or aged adult BM measured in the total number of viable cells present in the BM aspirate obtained at each time point assessed. Values indicate the geometric mean of pooled values from all mice in each group assessed in 2 independent
Figure 2 Similar in vivo repopulating activity of neonatal and adult CD49f + cells when cotransplanted with CD34 + CB ' helper ' cells. (A) Design of a cotransplantation strategy to compare the regenerative potential of CD49f + cells isolated from donors of different ages. (B) Flow-cytometric analysis of the cells in a representative BM aspirate obtained from a mouse that had undergone cotransplantation 12 weeks earlier with CB CD49f + cells and GFP-labeled CD34 + CB cells. (C-D) Percentage of human CD49f + cell (GFP -) (C), and NM, B-lymphoid, erythroid, and CD34 + progeny (D) from input CB, young adult BM, or aged adult BM measured in the total number of viable cells present in the BM aspirate obtained at each time point assessed. Values indicate the geometric mean of pooled values from all mice in each group assessed in 2 independentCC-BY-NC-ND · From the PDF
Figure 3 GF-stimulated CD49f + cells display a progressive age-related delay in vitro . (A) Design of the culture system used to compare the survival and cell division kinetics of CD49f + cells isolated from differently aged donors over 7 days of culture. (B) Kaplan-Meier curves of CD49f + cell survival in the cultures described in panel A (log-rank test, *** P < .001). (C) Weighted dose-response curves describing the completion of a fi rst division of the different sources of CD49f + tested. Curves re fl ect only CD49f + cells that remained viable past a fi rst division, or until the end of the assay without dividing. Error bars are indicated at the median point of each curve (CB = 57 hours, young adult = 75 hours, aged adult = 90 hours) and indicate the range de fi ned by ± 1 SEM of this estimate (*** P < .001). Time required for clones to complete a second (D) or third (E) division after completing their previous division (*** P < .001).
Figure 3 GF-stimulated CD49f + cells display a progressive age-related delay in vitro . (A) Design of the culture system used to compare the survival and cell division kinetics of CD49f + cells isolated from differently aged donors over 7 days of culture. (B) Kaplan-Meier curves of CD49f + cell survival in the cultures described in panel A (log-rank test, *** P < .001). (C) Weighted dose-response curves describing the completion of a fi rst division of the different sources of CD49f + tested. Curves re fl ect only CD49f + cells that remained viable past a fi rst division, or until the end of the assay without dividing. Error bars are indicated at the median point of each curve (CB = 57 hours, young adult = 75 hours, aged adult = 90 hours) and indicate the range de fi ned by ± 1 SEM of this estimate (*** P < .001). Time required for clones to complete a second (D) or third (E) division after completing their previous division (*** P < .001).CC-BY-NC-ND · From the PDF
Figure 4 Paired cell cycle state and molecular analysis of individually tracked primitive human hematopoietic cells reveal an age-associated prolongation of G1 in the adult cells. (A) Experimental design to enable the in vitro serial immuno fl uorescence tracking of changes in the properties of individual cells isolated from donors of different ages over time. (B) Representative fl uorescence images obtained from the experimental design described in panel A; scale bar = 100 μ m. (C) UMAP distribution of different phenotypic properties of individually assessed CD49f + and CD34 + CD38 -cells from CB and young or aged adult BM cultured for 0, 24, 42, 50, or 64 hours and analyzed as described in panel A. Assignment of regions of the UMAP distribution to the different phases of the cell cycle by K-means clustering (bottom-right). (D) Proportion of CD49f +
Figure 4 Paired cell cycle state and molecular analysis of individually tracked primitive human hematopoietic cells reveal an age-associated prolongation of G1 in the adult cells. (A) Experimental design to enable the in vitro serial immuno fl uorescence tracking of changes in the properties of individual cells isolated from donors of different ages over time. (B) Representative fl uorescence images obtained from the experimental design described in panel A; scale bar = 100 μ m. (C) UMAP distribution of different phenotypic properties of individually assessed CD49f + and CD34 + CD38 -cells from CB and young or aged adult BM cultured for 0, 24, 42, 50, or 64 hours and analyzed as described in panel A. Assignment of regions of the UMAP distribution to the different phases of the cell cycle by K-means clustering (bottom-right). (D) Proportion of CD49f +CC-BY-NC-ND · From the PDF
Figure 5 CD49f + cells show an aging-related increase in their 3GF concentration requirement to stimulate their division. (A) Weighted 3GF dose-response curves for CD49f + cell survival (top) and proliferation (bottom) monitored over a 7-day period in vitro as described in Figure 3A. Proliferation curves are normalized to cells that survived either past a fi rst division or for 7 days without dividing. Points indicate the mean ± 1 SEM of the survival and proliferation responses of each group of cell types assessed (* P < .05; *** P < .001). The 50% effective dose concentrations were interpolated from the fi tted curves (survival: CB = 0.11%, young adult = 0.09%, aged adult = 0.19%; proliferation: CB = 0.2%, young adult = 0.6%, aged adult = 0.9%). (B) Percent of clones that completed the indicated number of divisions by day 7 at 100% 3GF (left) or 1% 3GF (right). Lines connect the observed mean ± 1 SEM of the proportion of cells from each donor source that had completed the indicated number of divisions within 7 days. (*** P < .001; pairwise Kruskal-Wallis tests). (C) Kinetics of 3GF-stimulated timing of a fi rst division of CD49f + cells from different sources exposed to 100% (solid lines) or 1%
Figure 5 CD49f + cells show an aging-related increase in their 3GF concentration requirement to stimulate their division. (A) Weighted 3GF dose-response curves for CD49f + cell survival (top) and proliferation (bottom) monitored over a 7-day period in vitro as described in Figure 3A. Proliferation curves are normalized to cells that survived either past a fi rst division or for 7 days without dividing. Points indicate the mean ± 1 SEM of the survival and proliferation responses of each group of cell types assessed (* P < .05; *** P < .001). The 50% effective dose concentrations were interpolated from the fi tted curves (survival: CB = 0.11%, young adult = 0.09%, aged adult = 0.19%; proliferation: CB = 0.2%, young adult = 0.6%, aged adult = 0.9%). (B) Percent of clones that completed the indicated number of divisions by day 7 at 100% 3GF (left) or 1% 3GF (right). Lines connect the observed mean ± 1 SEM of the proportion of cells from each donor source that had completed the indicated number of divisions within 7 days. (*** P < .001; pairwise Kruskal-Wallis tests). (C) Kinetics of 3GF-stimulated timing of a fi rst division of CD49f + cells from different sources exposed to 100% (solid lines) or 1%CC-BY-NC-ND · From the PDF
Figure 6 AKT signaling drives the 3GF-activated survival and division of primitive human hematopoietic cells. (A) Experimental design used to compare the 3GF-stimulated time course of signaling protein activation assessed by fl ow cytometric analysis. (B) Median relative shift in the intensity of the indicated marker from the corresponding unstimulated sample in CD49f + cells exposed to 3GF for different times. Asterisks indicate signi fi cant differences from the unstimulated samples (* P < .05; ** P < .01; *** P < .001; bootstrapped probabilities). Paired survival (C) and proliferation (D) effects of dimethyl sulfoxide (d)- or triciribine (t)-mediated inhibition of AKT activation. (E) Model of CD49f + pathway activation after short-term 3GF stimulation. The length of the colored bars indicates the relative strength of activation of the indicated marker. CB responses are shown in navy, young adult in red, and aged adult in teal. (F) A model of the role of AKT signaling and other factors in regulating different stages of primitive human hematopoietic cell survival and proliferation.
Figure 6 AKT signaling drives the 3GF-activated survival and division of primitive human hematopoietic cells. (A) Experimental design used to compare the 3GF-stimulated time course of signaling protein activation assessed by fl ow cytometric analysis. (B) Median relative shift in the intensity of the indicated marker from the corresponding unstimulated sample in CD49f + cells exposed to 3GF for different times. Asterisks indicate signi fi cant differences from the unstimulated samples (* P < .05; ** P < .01; *** P < .001; bootstrapped probabilities). Paired survival (C) and proliferation (D) effects of dimethyl sulfoxide (d)- or triciribine (t)-mediated inhibition of AKT activation. (E) Model of CD49f + pathway activation after short-term 3GF stimulation. The length of the colored bars indicates the relative strength of activation of the indicated marker. CB responses are shown in navy, young adult in red, and aged adult in teal. (F) A model of the role of AKT signaling and other factors in regulating different stages of primitive human hematopoietic cell survival and proliferation.CC-BY-NC-ND · From the PDF

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Metadata

Authors
Hammond, C. A., Wu, S. W., Wang, F., MacAldaz, M. E., & Eaves, C. J.
Affiliations
1Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada
Venue
Blood
Publisher
American Society of Hematology
Published
2023-04-20
Volume
141
Issue
16
Pages
1990-2002
ISSN
  • 0006-4971
  • 1528-0020
DOI
10.1182/blood.2022017174
Cited by
40
References
12
Language
en
Licence
creativecommons.org/licenses/by-nc-nd/4.0/ (vor)

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Hammond, C. A., Wu, S. W., Wang, F., MacAldaz, M. E., & Eaves, C. J. (2023). Aging alters the cell cycle control and mitogenic signaling responses of human hematopoietic stem cells. Blood. https://doi.org/10.1182/blood.2022017174