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. 2010 May 20;115(20):4030-8.
doi: 10.1182/blood-2009-09-241000. Epub 2010 Mar 30.

AKT1 and AKT2 maintain hematopoietic stem cell function by regulating reactive oxygen species

Marisa M Juntilla  1 , Vineet D Patil, Marco Calamito, Rohan P Joshi, Morris J Birnbaum, Gary A Koretzky
Affiliations

AKT1 and AKT2 maintain hematopoietic stem cell function by regulating reactive oxygen species

Marisa M Juntilla et al. Blood. .

Abstract

Although AKT is essential for multiple cellular functions, the role of this kinase family in hematopoietic stem cells (HSCs) is unknown. Thus, we analyzed HSC function in mice deficient in the 2 isoforms most highly expressed in the hematopoietic compartment, AKT1 and AKT2. Although loss of either isoform had only a minimal effect on HSC function, AKT1/2 double-deficient HSCs competed poorly against wild-type cells in the development of myeloid and lymphoid cells in in vivo reconstitution assays. Serial transplantations revealed an essential role for AKT1 and AKT2 in the maintenance of long-term HSCs (LT-HSCs). AKT1/2 double-deficient LT-HSCs were found to persist in the G(0) phase of the cell cycle, suggesting that the long-term functional defects are caused by increased quiescence. Furthermore, we found that the intracellular content of reactive oxygen species (ROS) is dependent on AKT because double-deficient HSCs demonstrate decreased ROS. The importance of maintaining ROS for HSC differentiation was shown by a rescue of the differentiation defect after pharmacologically increasing ROS levels in double-deficient HSCs. These data implicate AKT1 and AKT2 as critical regulators of LT-HSC function and suggest that defective ROS homeostasis may contribute to failed hematopoiesis.

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Figures

Figure 1
Figure 1
Quantification of AKT1−/− or AKT2−/− HSCs in adult mice and assessment of function in serial transplantation. Total number of LSKs and LT-HSCs (CD150+CD48−LSKs) in bone marrow from femurs and tibias in (A) 3-month-old or (B) 12-month-old single knockout mice. Graphs represent the mean ± SEM in (A) n = 6 and (B) n = 4. (C) Percentage of single knockout (CD45.2+Ly5B6+) cells in the bone marrow LSK population 16 weeks after the initial transplantation (primary) and then 16 weeks after each serial transplantation (secondary and tertiary). FL cells from single-knockout fetuses (CD45.2+Ly5B6+) were mixed with wild-type FL cells (CD45.1+ B6.Ly5SJL) in a 3:1 (CD45.2+:CD45.1+) ratio and serially transplanted every 16 weeks. Bar graphs represent the mean ± SEM (n = 4).
Figure 2
Figure 2
Central and peripheral hematopoietic reconstitution by AKT1−/−AKT2−/− FL-derived HSCs. The frequency of (A) LSK cells in the lineage-negative gate or (B) LT-HSCs (CD150+CD48−LSKs) in the total LSK gate of E14.5 FLs. Graphs represent the mean ± SEM (n = 5). (C) The total number of colonies generated 10 days after equal numbers of FL cells were plated in methylcellulose media. The diversity of colonies was not different between the 2 genotypes. Data are represented as the percentage of wild-type colonies generated within each experiment and represent the mean ± SEM (n = 4). The frequency of (D) LSK cells in the lineage-negative gate or (E) LT-HSCs (CD150+CD48−LSKs) in the total LSK gate of bone marrow from irradiated mice reconstituted 12 weeks previously with FL cells of the indicated genotype. Graphs represent the mean ± SEM (n = 3). (F) The total number of cells in the splenic myeloid (Mac-1+Gr-1+), B (B220+), and T (TCRβ+) lineages in mice from panels D and E. Graphs represent the mean ± SEM (n = 3).
Figure 3
Figure 3
Trilineage reconstitution by AKT1−/−AKT2−/− HSCs in long-term and serial competitive transplantations. (A) The number of methylcellulose colonies generated from equal numbers of LTC-ICs. LTC-ICs were derived by culturing sorted LSK cells for 2 weeks on OP9 monolayers. Graph represents the mean ± SEM (n = 3) of the number of colonies as a percentage of the control (AKT1+/+AKT2+/−) for each experiment. (B) The percentage of AKT1−/−AKT2−/− (CD45.2+Ly5B6+) cells in the peripheral blood myeloid (Mac-1+Gr-1+), B (B220+), and T (TCRβ+) lineages 4, 6, 12, and 22 weeks after reconstitution. Graphs represent the mean ± SEM (n = 3) for each time point. (C) The percentage of AKT1−/−AKT2−/− (CD45.2+Ly5B6+) cells in the LSK population 16 weeks after the initial transplantation (primary) and after 2 additional 16-week serial transplantations (secondary and tertiary). The graph represents the mean ± SEM (n = 4) of the percentage of CD45.2+Ly5B6+ cells in the LSK population. The AKT1+/+AKT2+/− controls are the same samples represented in Figure 1C. (D) The percentage of AKT1−/−AKT2−/− (CD45.2+Ly5B6+) cells in the bone marrow LSK subset and the splenic myeloid (Mac1+Gr1+), B (B220+), and T (TCRβ+) subsets 16 weeks after the secondary transplantation in the mice from panel C (secondary time point). The graph represents the mean ± SEM (n = 4). The LSK data are the same as in panel C (secondary time point). (E) Splenic γδT-cell competitiveness in the absence of AKT1 and AKT2. γδT cells were identified by gating on the singlet, DAPI−, Thy1+, γδTCR+ population. LSK data are the same as the primary transplantation in panel C. The graph represents the mean ± SEM (n = 4).
Figure 4
Figure 4
AKT1 and AKT2 are not required for the development of myeloid precursor populations in vivo. (A) Gating scheme used to identify the GMP, CMP, and MEP populations present in the bone marrow of mice that were analyzed 16 weeks after the secondary serial bone marrow transplantation. The populations in panel A are gated on the singlet, DAPI-negative, lineage-negative population (left) and the singlet, DAPI-negative, lineage-negative, c-Kit+ population (right). (B) The percentage of CD45.2+Ly5B6+ cells present in the LSK, CMP, GMP, and MEP populations from the mice described in panel A. LSK data are the same as the secondary transplantation in Figure 3C. The graph represents the mean ± SEM (n = 4).
Figure 5
Figure 5
Generation of LT-HSCs and MPPs in a competitive environment. (A) The percentage of AKT1−/−AKT2−/− (CD45.2+Ly5B6+) cells in the BM LSK subset, and the splenic myeloid (Mac-1+Gr-1+), B (B220+), and T (TCRβ+) lineages 6 weeks after reconstitution. Graphs represent the mean ± SEM (n = 3). (B) The gating strategy used to generate the data in panels A, C, and D. Plots are representative of one experiment 6 weeks after reconstitution. (C-D) The percentage of AKT1−/−AKT2−/− (CD45.2+Ly5B6+) cells in the LSK, LT-HSC (CD150+CD48−LSK), or MPP (CD150–CD48−LSK) populations in the bone marrow of mice at either (C) 6 weeks or (D) 12 weeks after reconstitution. Graphs represent the mean ± SEM (n = 3 for panel C and n = 5 for panel D). In panels A through D, FL cells from AKT1−/−AKT2−/− or littermate control animals (CD45.2+Ly5B6+) were mixed with wild-type competitor FL cells (CD45.1+B6.Ly5SJL) in a 1:1 ratio and injected into lethally irradiated CD45.1+B6.Ly5SJL recipients. The LSK data depicted in panels A and C are the same.
Figure 6
Figure 6
Proliferation and apoptosis in AKT1−/−AKT2−/− LT-HSCs and MPPs. (A-B) The frequency of cells in the G0, G1, and S/G2/M phases of the cell cycle based on RNA (Pyronin Y) and DNA (Hoechst) content. The data in panels A and B are gated on the CD45.2+ LT-HSC (CD150+CD48−LSK) population. The graphs in panel B represent the mean ± SEM (n = 3). (C-D) The frequency of annexin V+ DAPI− cells in the CD45.2+ LT-HSC (CD150+CD48−LSK) or CD45.2+ MPP (CD150−CD48−LSK) compartment. The plots in panel C are gated on the CD45.2+ MPP (CD150−CD48−LSK) population and represent one experiment; the graphs in panel D represent the mean ± SEM (n = 3). Panels A through D are derived from mice reconstituted 12 weeks previously with wild-type (CD45.1+B6.Ly5SJL) and AKT1−/−AKT2−/− or AKT1+/+AKT2+/− FL-HSCs (CD45.2+Ly5B6+).
Figure 7
Figure 7
Intracellular ROS in AKT1−/−AKT2−/− LSK cells in vivo and their colony-forming capacity in vitro after pharmacologic increase of ROS. (A-B) ROS content of AKT1−/−AKT2−/− LSK cells. The histogram (A) represents one experiment, and the bar graph (B) represents the mean ± SEM (n = 5) of the MFI of the DCF-DA–treated cells normalized to the HBSS-only–treated cells (no DCF-DA) from each experiment. LSK cells were sorted from bone marrow chimeras, incubated with DCF-DA, and analyzed by flow cytometry. (C-D) ROS content in DN3 thymocytes. Thymocytes from 16-week primary competitive bone marrow chimeras were surface stained with CD4, CD8, CD25, and CD45.2. DN3 staged thymocytes were identified by CD4−CD8−CD25+ phenotype. The histogram (C) represents one experiment, and the bar graph (D) represents the mean ± SEM (n = 3) of the DCF-DA MFI of CD45.2+ cells divided by the DCF-DA MFI of CD45.1+ cells in the same tube. (E) Colony-forming capacity of LSK cells treated with BSO. The graphs represent the total number of colonies found in each culture as a percentage of those generated in the absence of BSO (no BSO = 1) and represent the mean ± SEM (n = 5). Equal numbers of freshly sorted LSK cells were plated in methylcellulose cultures with increasing concentrations of BSO for 10 days before analysis. The number of colonies per plate (mean ± SEM) that used AKT1+/+AKT2+/− cells was 0μM BSO 45 ± 3, 0.0167μM BSO 34 ± 3, 0.020μM BSO 26 ± 2, 0.025μM BSO 29 ± 1, 0.050μM BSO 15 ± 2, and 0.100μM BSO no growth and for AKT1−/−AKT2−/− cells was 0μM BSO 32 ± 2, 0.0167μM BSO 38 ± 1, 0.020μM BSO 32 ± 3, 0.025μM BSO 39 ± 3, 0.050μM BSO 23 ± 2, and 0.100μM BSO 3 ± 1. (F) Effect of BSO treatment on LTC-ICs. LSK cells were grown in LTC-IC cultures for 2 weeks in the presence of 0.025μM BSO. Then, equal numbers of cells were harvested from the LTC-IC cultures and plated in methylcellulose in the absence of BSO for 10 days. The graphs represent the total number of colonies found in each methylcellulose culture as a percentage of those generated in the absence of BSO (no BSO = 1) and represent the mean ± SEM (n = 3).
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