Pharmacological inhibition of AKT activity in human CD34+ cells enhances their ability to engraft immunodeficient mice
2016; Elsevier BV; Volume: 45; Linguagem: Inglês
10.1016/j.exphem.2016.09.003
ISSN1873-2399
AutoresSisi Chen, Rui Gao, Michihiro Kobayashi, Hao Yu, Chonghua Yao, Reuben Kapur, Mervin C. Yöder, Yan Liu,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
Resumo•Inhibition of AKT increases human CD34+ cell quiescence.•Inhibition of AKT enhances the colony formation potential of human CD34+ cells.•Pharmacological inhibition of AKT enhances the engraftment of HSPCs in vivo. Although practiced clinically for more than 40 years, the use of hematopoietic stem cell (HSC) transplantation remains limited by the inability to expand functional HSCs ex vivo. To determine the role of phosphoinositide 3-kinase (PI3K)/AKT signaling in human hematopoietic stem and progenitor cell (HSPC) maintenance, we examined the effect of genetic and pharmacological inhibition of AKT on human umbilical cord blood (UCB) CD34+ cells. We found that knock-down of AKT1 in human UCB CD34+ cells using short interfering RNAs targeting AKT1 enhances their quiescence and colony formation potential in vitro. We treated human UCB CD34+ cells with an AKT-specific inhibitor (AKTi) and performed both in vitro and in vivo stem and progenitor cell assays. We found that ex vivo treatment of human HSPCs maintains CD34 expression and enhances colony formation in serial replating assays. Moreover, pharmacological inhibition of AKT enhances the short-term repopulating potential of human UCB CD34+ cells in immunodeficient mice. Mechanistically, genetic and pharmacological inhibition of AKT activity promotes human HSPC quiescence. These preclinical results suggest a positive role for AKTi during ex vivo culture of human UCB HSPCs. Although practiced clinically for more than 40 years, the use of hematopoietic stem cell (HSC) transplantation remains limited by the inability to expand functional HSCs ex vivo. To determine the role of phosphoinositide 3-kinase (PI3K)/AKT signaling in human hematopoietic stem and progenitor cell (HSPC) maintenance, we examined the effect of genetic and pharmacological inhibition of AKT on human umbilical cord blood (UCB) CD34+ cells. We found that knock-down of AKT1 in human UCB CD34+ cells using short interfering RNAs targeting AKT1 enhances their quiescence and colony formation potential in vitro. We treated human UCB CD34+ cells with an AKT-specific inhibitor (AKTi) and performed both in vitro and in vivo stem and progenitor cell assays. We found that ex vivo treatment of human HSPCs maintains CD34 expression and enhances colony formation in serial replating assays. Moreover, pharmacological inhibition of AKT enhances the short-term repopulating potential of human UCB CD34+ cells in immunodeficient mice. Mechanistically, genetic and pharmacological inhibition of AKT activity promotes human HSPC quiescence. These preclinical results suggest a positive role for AKTi during ex vivo culture of human UCB HSPCs. Several decades of successful bone marrow transplantations have demonstrated the therapeutic importance of hematopoietic stem cells (HSCs) [1Walasek M.A. van Os R. de Haan G. 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Hematopoietic stem cell expansion: challenges and opportunities.Ann N Y Acad Sci. 2012; 1266: 138-150Crossref PubMed Scopus (159) Google Scholar, 2Gluckman E. History of cord blood transplantation.Bone Marrow Transplant. 2009; 44: 621-626Crossref PubMed Scopus (122) Google Scholar, 3Wagner J.E. Gluckman E. Umbilical cord blood transplantation: the first 20 years.Semin Hematol. 2010; 247: 3-12Abstract Full Text Full Text PDF Scopus (81) Google Scholar, 4Hofmeister C.C. Zhang J. Knight K.L. Le P. Stiff P.J. Ex vivo expansion of umbilical cord blood stem cells for transplantation: growing knowledge from the hematopoietic niche.Bone Marrow Transplant. 2007; 39: 11-23Crossref PubMed Scopus (186) Google Scholar]. However, low cell numbers in single UCB units have limited the suitability of UCB transplantation for adult patients [1Walasek M.A. van Os R. de Haan G. Hematopoietic stem cell expansion: challenges and opportunities.Ann N Y Acad Sci. 2012; 1266: 138-150Crossref PubMed Scopus (159) Google Scholar, 2Gluckman E. History of cord blood transplantation.Bone Marrow Transplant. 2009; 44: 621-626Crossref PubMed Scopus (122) Google Scholar, 3Wagner J.E. Gluckman E. Umbilical cord blood transplantation: the first 20 years.Semin Hematol. 2010; 247: 3-12Abstract Full Text Full Text PDF Scopus (81) Google Scholar, 4Hofmeister C.C. Zhang J. Knight K.L. Le P. Stiff P.J. Ex vivo expansion of umbilical cord blood stem cells for transplantation: growing knowledge from the hematopoietic niche.Bone Marrow Transplant. 2007; 39: 11-23Crossref PubMed Scopus (186) Google Scholar]. Methods to increase robustly the number of cells that give a rapid and sustained blood count recovery would enable the use of UCB in more patients [1Walasek M.A. van Os R. de Haan G. Hematopoietic stem cell expansion: challenges and opportunities.Ann N Y Acad Sci. 2012; 1266: 138-150Crossref PubMed Scopus (159) Google Scholar, 2Gluckman E. History of cord blood transplantation.Bone Marrow Transplant. 2009; 44: 621-626Crossref PubMed Scopus (122) Google Scholar, 3Wagner J.E. Gluckman E. Umbilical cord blood transplantation: the first 20 years.Semin Hematol. 2010; 247: 3-12Abstract Full Text Full Text PDF Scopus (81) Google Scholar, 4Hofmeister C.C. Zhang J. Knight K.L. Le P. Stiff P.J. Ex vivo expansion of umbilical cord blood stem cells for transplantation: growing knowledge from the hematopoietic niche.Bone Marrow Transplant. 2007; 39: 11-23Crossref PubMed Scopus (186) Google Scholar]. 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Transient silencing of PTEN in human CD34(+) cells enhances their proliferative potential and ability to engraft immunodeficient mice.Exp Hematol. 2012; 40: 84-91Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 23Rohrabaugh S.L. Campbell T.B. Hangoc G. Broxmeyer H.E. Ex vivo rapamycin treatment of human cord blood CD34+ cells enhances their engraftment of NSG mice.Blood Cells Mol Dis. 2011; 46: 318-320Crossref PubMed Scopus (31) Google Scholar]. Although transient silencing of PTEN in human CD34+ cells enhances their proliferation potential and short-term repopulation capability [22Kim I. Kim Y.J. Métais J.Y. Dunbar C.E. Larochelle A. Transient silencing of PTEN in human CD34(+) cells enhances their proliferative potential and ability to engraft immunodeficient mice.Exp Hematol. 2012; 40: 84-91Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar], ex vivo rapamycin treatment of human UCB CD34+ cells, which inhibits mTOR activity, enhances their engraftment of immunodeficient mice in serial transplantation assays [23Rohrabaugh S.L. Campbell T.B. Hangoc G. Broxmeyer H.E. Ex vivo rapamycin treatment of human cord blood CD34+ cells enhances their engraftment of NSG mice.Blood Cells Mol Dis. 2011; 46: 318-320Crossref PubMed Scopus (31) Google Scholar]. Given that rapamycin can induce feedback activation of AKT signaling through an insulin-like growth factor-1 receptor-dependent mechanism [24Zoncu R. Efeyan A. 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In this study, we found that knock-down of AKT1 in human UCB CD34+ cells using small interfering RNAs (siRNAs) targeting AKT1 enhances their quiescence and colony formation potential in vitro. Importantly, we discovered that pharmacological inhibition of AKT activity with an AKT-specific inhibitor (AKTi) in human UCB CD34+ cells promotes their quiescence and enhances their engraftment in immunodeficient mice. Our studies may facilitate the development of innovative clinical strategies that can enhance the engraftment of human UCB HSPCs. For the repopulation assay, 6- to 8-week-old NSG (NOD.Cg-Prkdscid IL2rgtm1Wj1/Sz) mice were obtained from an on-site core breeding colony. The institutional animal care and use committee of the Indiana University School of Medicine approved all experimental procedures. Cells were washed twice with ice-cold phosphate-buffered saline and lysed on ice in radioimmunoprecipitation assay buffer (Sigma-Aldrich) supplemented with protease inhibitor (Roche) and phosphatase inhibitor (Roche). Cell lysates were sonicated and centrifuged at 13,200 rpm for 10 min; boiled with lithium dodecyl sulfate sample buffer (ThermoFisher Scientific); separated by NuPAGE gel (ThermoFisher Scientific); transferred electrophoretically to a polyvinylidene fluoride membrane (EMD Millipore); and immunoblotted with phospho-AKT (Ser473), phospho-AKT (Thr308), AKT, and GAPDH (Cell Signaling Technology), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). The blots were developed using the enhanced chemiluminescence technique with SuperSignal West Femto Maximum Sensitivity Chemiluminescent Substrate (Fisher Scientific). Human HSPCs were identified and evaluated by flow cytometry using a single-cell suspension stained with antibodies against human CD34-fluorescein isothiocyanate (FITC), human CD34-phycoerythrin (PE), human CD3-FITC (BD Biosciences), GlyA (human CD235a)-PE (eBioscience), human CD71-allophycocyanin (APC), human CD34-PE/CY7, human CD3-APC/CY7, human CD45-APC, human CD19-PerCP-Cy5.5, human CD14-PE/CY7, and human CD33-FITC (BioLegend). Experiments were performed on Accuri C6 and FACSLSR II cytometers (BD Biosciences) and analyzed with FlowJo version 9.3.3 software (TreeStar). Normal human UCB samples were collected with institutional approval. Mononuclear cells (MNCs) were isolated by Ficoll-Paque Plus density gradient centrifugation (1.077 g/mL, GE Healthcare Life Sciences). CD34+ cells were purified from MNCs by positive selection using the human CD34 MicroBead Kit and LS separation columns (Miltenyi Biotec) according to the manufacturer's instructions. A total of 40,000 purified CD34+ cells were expanded in StemSpan Serum-Free Expansion Medium (STEMCELL Technologies) supplemented with recombinant human (rh) SCF (100 ng/mL), rhTPO (100 ng/mL), and rh-Flt-3L (100 ng/mL). To differentiate human HSPCs, purified CD34+ cells were cultured in myeloid-promoting conditions (SCF, 100 ng/mL; FLT-3 ligands, 10 ng/mL; IL-3, 20 ng/mL; IL-6, 20 ng/mL; granulocyte-macrophage colony-stimulating factor, 20 ng/mL; and granulocyte colony-stimulating factor, 20 ng/mL) and in erythroid-promoting conditions (EPO, 6 IU/mL; SCF, 100 ng/mL). Cytokines were from PeproTech. AKTi was from Merck [26She Q.B. Chandarlapaty S. Ye Q. et al.Breast tumor cells with PI3K mutation or HER2 amplification are selectively addicted to Akt signaling.PLoS One. 2008; 3: e3065Crossref PubMed Scopus (237) Google Scholar]. Purified CD34+ cells were stained with 2.5 μmol/L Hoechst 33342 (Molecular Probes) for 45 minutes at 37°C. Pyronin Y was added at a final concentration of 1 μmol/L and cells were incubated for a further 45 minutes at 37°C. Thereafter, cells were kept on ice for 30 minutes. Cells were washed twice and stained with APC-conjugated antihuman CD34 antibody [27Miyata Y. Liu Y. Jankovic V. et al.Cyclin C regulates human hematopoietic stem/progenitor cell quiescence.Stem Cells. 2010; 28: 308-317PubMed Google Scholar]. Hematopoietic clonogenic progenitors were determined in methylcellulose medium (MethoCult H4435, StemCell Technologies) using 1,000 cultured or 250 sorted CD34+ cells per well (six-well plate). Colonies were scored 14 days after plating. For secondary CFC assays, cells were harvested and washed with phosphate-buffered saline on day 14 after the first plating and 1 × 104 cells were replated as described above. Colonies were scored at day 14. NSG mice were from the Indiana University In Vivo Therapeutic Core Facility. At the age of 8 weeks, mice were irradiated with a single dose of 2.5 Gy and then 200,000 human CD34+ cells were injected into irradiated NSG mice via their tail vein. Peripheral blood of NSG mice was obtained every 4 weeks after transplantation, stained with anti-human CD45 FITC and anti-mouse CD45 PE antibodies, and then analyzed by flow cytometry. After 12 weeks, bone marrow cells were harvested and analyzed for human CD45 expression to define the engraftment efficiency. Accell nontargeting pool control siRNA (control siRNAs) and Accell human AKT1 smart pool siRNA (AKT1 siRNAs) were obtained from GE Dharmacon. A total of 20,000 human UCB CD34+ cells were suspended in Accell Delivery Medium (Dharmacon) with cytokines and seeded in 96-well plate. siRNAs were added in a final concentration of 1 μmol/L according to the manufacturer's instructions (Dharmacon). Cells were transferred to CD34+ basic medium 48 hours after transfection [28Bonifazi P. D'Angelo C. Zagarella S. et al.Intranasally delivered siRNA targeting PI3K/Akt/mTOR inflammatory pathways protects from aspergillosis.Mucosal Immunol. 2010; 3: 193-205Crossref PubMed Scopus (61) Google Scholar]. Sorted CD34+ cells were plated in eight-well chamber slides. Senescent cells were detected with the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology) according to the manufacturer's instructions [29Smith L.L. Yeung J. Zeisig B.B. et al.Functional crosstalk between Bmi1 and MLL/Hoxa9 axis in establishment of normal hematopoietic and leukemic stem cells.Cell Stem Cell. 2011; 8: 649-662Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar]. Data were analyzed statistically using either the Student t test or one-way ANOVA (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant). To determine whether knock-down of AKT1 expression alters the behavior of HSPCs, we transiently transfected either control or AKT1 siRNAs into purified CD34+ cells. AKT1 mRNA expression was decreased by 50% in AKT1 siRNA-transfected cells at 48 hours compared with control siRNA-transfected cells (Fig. 1A). To determine how transient knock-down of AKT1 affects the primitiveness of human HSPCs, we examined the proliferative capacity of CD34+ cells in early-acting cytokine (rhSCF, rhTPO, and rh-Flt-3L)-containing liquid cultures after transfection of control and AKT1 siRNAs. We quantified CD34 expression by fluorescence-activated cell sorting (FACS) over time and found that AKT1 deficiency altered neither the frequency nor the absolute number of CD34+ cells (Fig. 1B and 1C). We then examined the effect of AKT1 deficiency on HSPC quiescence using pyronin Y and Hoechst 33342 staining and found that transient knock-down of AKT1 was sufficient to increase human HSPC quiescence (Fig. 1D and 1E). To determine whether genetic inhibition of AKT affects CD34+ cell survival and senescence, we assessed the apoptosis and senescence of CD34+ cells 7 days after transfection. We observed a comparable level of apoptotic [Annexin V+/7-amino-actinomycin D-negative (7-AAD−)] cells in both control siRNA- and AKT1 siRNA-transfected CD34+ cells (Fig. 1F). We found that knock-down of AKT1 did not induce senescence in CD34+ cells (data not shown). These findings suggest that transient knock-down of AKT1 induces neither apoptosis nor senescence in HSPCs. To examine the effect of knocking down AKT1 on HSPC function, we performed CFC assays. When we used 250 sorted CD34+ cells 7 days after transfection, we observed that AKT1 siRNA-transfected CD34+ cells formed more colonies compared with control siRNA-transfected CD34+ cells (Fig. 1G). Therefore, transient knock-down of AKT1 increases human HSPC quiescence and enhances their colony formation potential. We have shown previously that AKTi can inhibit AKT activation efficiently in both mouse and human HSPCs [21Liu Y. Liu F. Yu H. et al.Akt phosphorylates the transcriptional repressor Bmi1 to block its association with tumor suppressing Ink4a-Arf locus.Sci Signal. 2012; 5: ra77Crossref PubMed Scopus (37) Google Scholar, 30Vu L.P. Perna F. Wang L. et al.PRMT4 blocks myeloid differentiation by assembling a methyl-RUNX1-dependent repressor complex.Cell Rep. 2013; 5: 1625-1638Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar]. We found that treatment of CD34+ cells with AKTi (1 μmol/L) decreased AKT phosphorylation significantly at both serine 473 and threonine 308 (Fig. 2A), demonstrating that AKT activity was inhibited by AKTi treatment. To determine how AKTi affects the primitiveness of human HSPCs, we examined the proliferative capacity of CD34+ cells in early-acting cytokine (rhSCF, rhTPO, and rh-Flt-3L)-containing liquid cultures in the presence of AKTi (1 μmol/L). We quantified CD34 expression by FACS over time and found that a significantly greater proportion of AKTi-treated CD34+ cells retained CD34 expression (Fig. 2B and 2C). However, AKTi treatment did not increase the absolute number of CD34+ cells over time (Fig. 2D). We also examined the effect of AKT inhibition on primitive human HSCs (CD34+CD38−CD45RA−CD90+) and found that AKTi treatment did not increase the number of primitive HSCs (data not shown). Therefore, AKTi promotes the retention of CD34 expression in hematopoietic progenitor cells after ex vivo culture. We then performed CFC assays using 1,000 cells from dimethylsulfoxide (DMSO)- or AKTi-treated cultures at day 7 and found that AKTi-treated cells growing in liquid culture generated more colony-forming units (CFUs) than DMSO-treated cells during both primary and secondary CFC assays (Fig. 2E and 2F). When we used 250 sorted CD34+ cells 7 days after DMSO or AKTi (0.2 and 0.5 μmol/L, respectively) treatment in the CFC assays, we observed that AKTi-treated CD34+ cells formed more colonies compared with DMSO-treated CD34+ cells (Fig. 2G). Therefore, pharmacologic inhibition of AKT retains human HSPC primitiveness, leading to a greater capacity to generate CFUs. Quiescence is an important feature of human HSPCs [31Doulatov S. Notta F. Laurenti E. Dick J.E. Hematopoiesis: a human perspective.Cell Stem Cell. 2012; 10: 120-136Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar, 32Jetmore A. Plett P.A. Tong X. et al.Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34(+) cells transplanted into conditioned NOD/SCID recipients.Blood. 2002; 99: 1585-1593Crossref PubMed Scopus (89) Google Scholar, 33Srour E.F. Tong X. Sung K.W. et al.Modulation of in vitro proliferation kinetics and primitive hematopoietic potential of individual human CD34+CD38-/lo cells in G0.Blood. 2005; 105: 3109-3116Crossref PubMed Scopus (17) Google Scholar, 34Broxmeyer H.E. Orschell C.M. Clapp D.W. et al.Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist.J Exp Med. 2005; 201: 1307-1318Crossref PubMed Scopus (891) Google Scholar]. Given that knocking down AKT1 expression in human CD34+ cells enhances their quiescence (Fig. 1D and 1E), we speculated that pharmacological inhibition of AKT activity would promote human HSPC quiescence. To test this, we cultured UCB CD34+ cells in the presence or the absence of AKTi (0.1 or 1 μmol/L) for 7 days and then examined the frequency of quiescent CD34+ cells using pyronin Y and Hoechst 33342 staining. We found that both low (0.1 μmol/L) and high (1 μmol/L) concentrations of AKTi increased the frequency of quiescent CD34+ cells significantly compared with DMSO treatment (Fig. 3A and 3B), indicating that pharmacological inhibition of AKT activity promotes human HSPC quiescence. To determine whether inhibiting AKT activity affects CD34+ cell survival, we assessed the apoptosis of CD34+ cells 2 days after AKTi treatment and observed comparable level of apoptotic cells (Annexin V+/7-AAD−) in both DMSO- (0.01%) and AKTi (1 μmol/L)-treated CD34+ cells (Fig. 3C). We observed no increase in apoptotic CD34+ cells (Annexin V+/7-AAD−) 7 days after AKTi (0.1 or 1 μmol/L) treatment (Fig. 3D), suggesting that pharmacological inhibition of AKT activity does not induce apoptosis in HSPCs. To determine the effect of AKTi treatment on HSPC senescence, we performed senescence analysis using SA-β-gal staining [29Smith L.L. Yeung J. Zeisig B.B. et al.Functional crosstalk between Bmi1 and MLL/Hoxa9 axis in establishment of normal hematopoietic and leukemic stem cells.Cell Stem Cell. 2011; 8: 649-662Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar] and found that neither DMSO nor AKTi treatment induced senescence in human CD34+ cells (Fig. 3E). We used late-passage Bmi1−/− mouse embryonic fibroblasts, which undergo senescence, as positive controls [35Jacobs J.J. Kieboom K. Marino S. DePinho R.A. van Lohuizen M. The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus.Nature. 1999; 397: 164-168Crossref PubMed Scopus (1334) Google Scholar]. Therefore, AKT inhibition retains human HSPC primitiveness, at least in part through promoting quiescence. To investigate whether AKT activity is important for the differentiation of human HSPCs, we first placed purified CD34+ cells in myeloid-promoting medium along with DMSO (0.01%) or AKTi (1 μmol/L). The expression of the human myeloid marker CD11b was assessed by flow cytometry at day 9. The percentage of mature myeloid cells (CD11b+ cells) was significantly lower in the AKTi-treated group compared with DMSO-treated cells (Fig. 4A), revealing that myeloid differentiation of human CD34+ cells may be delayed upon AKT inhibition. We then analyzed the effect of AKT inhibition on erythroid differentiation by culturing purified CD34+ cells in erythroid-promoting medium along with DMSO (0.01%) or AKTi (1 μmol/L). The expression of human CD71 and GlyA was assessed by flow cytometry at day 5. The generation of mature erythroid precursor cells (CD71+/GlyA+ cells) was decreased significantly after AKTi treatment compared with DMSO-treated cells (Fig. 4B and 4C). These findings demonstrated that inhibition of AKT activity delays human HSPC differentiation toward the myeloid and erythroid lineages. Quiescent HSCs exhibit a significantly higher engraftment potential than proliferating HSCs in transplantation assays [32Jetmore A. Plett P.A. Tong X. et al.Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34(+) cells transplanted into conditioned NOD/SCID recipients.Blood. 2002; 99: 1585-1593Crossref PubMed Scopus (89) Google Scholar, 33Srour E.F. Tong X. Sung K.W. et al.Modulation of in vitro proliferation kinetics and primitive hematopoietic potential of individual human CD34+CD38-/lo cells in G0.Blood. 2005; 105: 3109-3116Crossref PubMed Scopus (17) Google Scholar, 34Broxmeyer H.E. Orschell C.M. Clapp D.W. et al.Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist.J Exp Med. 2005; 201: 1307-1318Crossref PubMed Scopus (891) Google Scholar]. Therefore, we sought to determine whether the enhanced quiescence seen in AKTi-treated CD34+ cells would improve their engraftability in immunodeficient mice. Whereas 1 μM AKTi treatment enhanced the colony formation potential of CD34+ cells (Fig. 2E–2G), inhibiting AKT activity decreased myeloid and erythroid differentiation (Fig. 4). Further, we found that CD34− cells showed increased apoptosis after 1 μmol/L AKTi treatment (data not shown). To avoid the potential negative impact of high concentrations of AKTi on transplantation outcome, we decided to use a low concentration of AKTi (0.1 μmol/L), which enhanced the colony formation potential as well as the quiescence of CD34+ cells (Figure 2, Figure 3), in transplantation experiments. An equal number of human CD34+ cells were treated with DMSO or AKTi (0.1 μmol/L) in vitro. Seven days later, we harvested cells and transplanted 200,000 cultured cells into sublethally irradiated NOD/SCID IL-2Rγ null mice. The engraftment of human cells in mice was analyzed every 4 weeks by monitoring human CD45 expression in peripheral blood harvested from the recipient mice. As shown in Figure 5A, we observed significantly increased engraftment in the AKTi-treated group compared with the control group at both 4 and 8 weeks after transplantation. Twelve weeks after transplantation, we examined human CD45 expression in the cells isolated from mouse bone marrow and observed a significant enhancement of AKTi-treated cell engraftment compared with DMSO-treated CD34+ cells (Fig. 5B). Next, we analyzed the multilineage reconstitution potential of AKTi-treated CD34+ cells in the bone marrow of recipient mice. Both the control group and the AKTi-treated group were capable of undergoing myeloid and lymphoid repopulation (Fig. 5C–5E). Whereas AKTi-treated CD34+ cells showed enhanced B-lineage reconstitution compared with the control group, inhibition of AKT activity decreased T-cell and myeloid differentiation of CD34+ cells (Fig. 5C–5E), which is consistent with the in vitro liquid culture experiment shown in Figure 4A and 4B. Therefore, AKT appears to restrict the self-renewal capacity of human HSPCs by promoting their cell-cycle entry and terminal differentiation and pharmacological inhibition of AKT activity leads to enhanced quiescence and better engraftability of human CD34+ cells. Clinical hematopoietic transplantation outcomes are strongly correlated with the numbers of cells infused, so low cell numbers in single UCB units have limited the suitability of UCB transplantation for adult patients [1Walasek M.A. van Os R. de Haan G. Hematopoietic stem cell expansion: challenges and opportunities.Ann N Y Acad Sci. 2012; 1266: 138-150Crossref PubMed Scopus (159) Google Scholar, 2Gluckman E. History of cord blood transplantation.Bone Marrow Transplant. 2009; 44: 621-626Crossref PubMed Scopus (122) Google Scholar, 3Wagner J.E. Gluckman E. Umbilical cord blood transplantation: the first 20 years.Semin Hematol. 2010; 247: 3-12Abstract Full Text Full Text PDF Scopus (81) Google Scholar]. The ability to increase the number of functional human HSCs in vitro or in vivo could provide improved treatment options for clinical transplantation and gene therapy protocols; however, clinically relevant expansion of HSCs has been difficult to achieve [1Walasek M.A. van Os R. de Haan G. Hematopoietic stem cell expansion: challenges and opportunities.Ann N Y Acad Sci. 2012; 1266: 138-150Crossref PubMed Scopus (159) Google Scholar, 2Gluckman E. History of cord blood transplantation.Bone Marrow Transplant. 2009; 44: 621-626Crossref PubMed Scopus (122) Google Scholar, 3Wagner J.E. Gluckman E. Umbilical cord blood transplantation: the first 20 years.Semin Hematol. 2010; 247: 3-12Abstract Full Text Full Text PDF Scopus (81) Google Scholar, 4Hofmeister C.C. Zhang J. Knight K.L. Le P. Stiff P.J. Ex vivo expansion of umbilical cord blood stem cells for transplantation: growing knowledge from the hematopoietic niche.Bone Marrow Transplant. 2007; 39: 11-23Crossref PubMed Scopus (186) Google Scholar]. In this study, we report that pharmacological inhibition of AKT activity in human UCB CD34+ cells enhances their ability to engraft immunodeficient mice through promoting HSPC quiescence. In addition, our work uncovered an important role of PI3K/AKT signaling in regulating human HSPC maintenance and lineage commitments. PI3K/PTEN/AKT signaling regulates murine HSC function because the constitutive activation of AKT depletes the HSC pool [17Manning B.D. Cantley L.C. AKT/PKB signaling: navigating downstream.Cell. 2007; 129: 1261-1274Abstract Full Text Full Text PDF PubMed Scopus (4571) Google Scholar, 18Zhang J. Grindley J.C. Yin T. et al.PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention.Nature. 2006; 441: 518-522Crossref PubMed Scopus (672) Google Scholar, 19Kharas M.G. Okabe R. Ganis J.J. et al.Constitutively active AKT depletes hematopoietic stem cells and induces leukemia in mice.Blood. 2010; 115: 1406-1415Crossref PubMed Scopus (217) Google Scholar]. In addition, we have demonstrated that AKT-mediated phosphorylation of Bmi1 inhibits HSC self-renewal [21Liu Y. Liu F. Yu H. et al.Akt phosphorylates the transcriptional repressor Bmi1 to block its association with tumor suppressing Ink4a-Arf locus.Sci Signal. 2012; 5: ra77Crossref PubMed Scopus (37) Google Scholar]. Based upon these observations, we hypothesized that suppressing PI3K/AKT signaling in human HSPCs promotes their quiescence and enhances their self-renewal capability. Using a serum-free culture system containing early-acting cytokines, we showed that pharmacological inhibition of AKT activity in UCB-derived CD34+ cells ex vivo improves human HSPC engraftment in NSG mice. Furthermore, inhibition of AKT activity promotes human HSPC quiescence. Transient knock-down of PTEN in human CD34+ cells enhances their proliferation, promoting quiescent HSPCs to enter the cell cycle [22Kim I. Kim Y.J. Métais J.Y. Dunbar C.E. Larochelle A. Transient silencing of PTEN in human CD34(+) cells enhances their proliferative potential and ability to engraft immunodeficient mice.Exp Hematol. 2012; 40: 84-91Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar]. Therefore, activation of PI3K/AKT signaling promotes human HSPC proliferation. Given that HSC quiescence is correlated with its self-renewal capability [32Jetmore A. Plett P.A. Tong X. et al.Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34(+) cells transplanted into conditioned NOD/SCID recipients.Blood. 2002; 99: 1585-1593Crossref PubMed Scopus (89) Google Scholar, 33Srour E.F. Tong X. Sung K.W. et al.Modulation of in vitro proliferation kinetics and primitive hematopoietic potential of individual human CD34+CD38-/lo cells in G0.Blood. 2005; 105: 3109-3116Crossref PubMed Scopus (17) Google Scholar, 34Broxmeyer H.E. Orschell C.M. Clapp D.W. et al.Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist.J Exp Med. 2005; 201: 1307-1318Crossref PubMed Scopus (891) Google Scholar], the enhanced engraftability of human HSPCs treated with AKT inhibitor is at least in part due to their enhanced quiescence. In addition to HSC maintenance, PTEN also regulates terminal differentiation [18Zhang J. Grindley J.C. Yin T. et al.PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention.Nature. 2006; 441: 518-522Crossref PubMed Scopus (672) Google Scholar]. Loss of PTEN promotes both myeloid and T-cell differentiation, but impairs B-cell production, in mice [18Zhang J. Grindley J.C. Yin T. et al.PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention.Nature. 2006; 441: 518-522Crossref PubMed Scopus (672) Google Scholar]. We found that pharmacological inhibition of AKT activity in human CD34+ cells results in decreased myeloid and T-cell differentiation in vivo. AKTi treatment also decreases erythroid differentiation of human HSPCs. Therefore, the PI3K/AKT signaling pathway promotes differentiation of human HSPCs. Cancer patients that receive chemotherapy normally develop neutropenia, increasing the risk of infections and resulting in treatment delay [36Yang B.B. Savin M.A. Green M. Prevention of chemotherapy-induced neutropenia with pegfilgrastim: pharmacokinetics and patient outcomes.Chemotherapy. 2012; 58: 387-398Crossref PubMed Scopus (30) Google Scholar, 37Mehdi I. Al Bahrani B. Chemotherapy-induced neutropenic necrotizing enterocolitis: a review.J Pak Med Assoc. 2012; 62: 718-723PubMed Google Scholar]. Neutrophils are essential for the innate immune response and can protect patients from bacterial infections in the initial phase of transplantation, especially for patients undergoing myelosuppressive chemotherapy [38Chotinantakul K. Prasajak P. Leeanansaksiri W. Wnt1 accelerates an ex vivo expansion of human cord blood CD34+CD38− cells.Stem Cells Int. 2013; 2013: 909812Crossref PubMed Scopus (8) Google Scholar, 39McNiece I. Jones R. Bearman S.I. et al.Ex vivo expanded peripheral blood progenitor cells provide rapid neutrophil recovery after high-dose chemotherapy in patients with breast cancer.Blood. 2000; 96: 3001-3007PubMed Google Scholar]. Therefore, the capability to generate matured blood cells is also critical for stem cell transplantation [11Delaney C. Heimfeld S. Brashem-Stein C. Voorhies H. Manger R.L. Bernstein I.D. Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution.Nat Med. 2010; 16: 232-236Crossref PubMed Scopus (596) Google Scholar, 40Shpall E.J. Quinones R. Giller R. et al.Transplantation of ex vivo expanded cord blood.Biol Blood Marrow Transplant. 2002; 8: 368-376Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar]. Although we observed increased engraftment of CD34+ cells treated with AKTi, the myeloid differentiation capacity of human HSPCs was decreased (Fig. 6). To compensate for the limitation of AKTi in human HSPC expansion, other factors, including Wnt activator [38Chotinantakul K. Prasajak P. Leeanansaksiri W. Wnt1 accelerates an ex vivo expansion of human cord blood CD34+CD38− cells.Stem Cells Int. 2013; 2013: 909812Crossref PubMed Scopus (8) Google Scholar, 41Willert K. Brown J.D. Danenberg E. et al.Wnt proteins are lipid-modified and can act as stem cell growth factors.Nature. 2003; 423: 448-452Crossref PubMed Scopus (1771) Google Scholar, 42Perry J.M. He X.C. Sugimura R. et al.Cooperation between both Wnt/{beta}-catenin and PTEN/PI3K/Akt signaling promotes primitive hematopoietic stem cell self-renewal and expansion.Genes Dev. 2011; 25: 1928-1942Crossref PubMed Scopus (135) Google Scholar], should be used in combination with AKTi. We expect that the combination of AKTi and the activator of canonical Wnt signaling will promote the ex vivo expansion of functional HSPCs without affecting their differentiation capability (Fig. 6). In summary, our work has identified an important role of PI3K/AKT signaling in regulating human HSPC maintenance. Our data suggest that modulating AKT activity in human HSPCs may be useful in enhancing their engraftability. Our findings may have a positive impact on the field of stem cell transplantation and provide novel clinical strategies that can enhance human HSPC transplantation outcome. The authors thank the In Vivo Therapeutics Core of the Indiana University Simon Cancer Center, the nursing staff and Dr. Arthur Baluyut at the St. Vincent Hospital (Indianapolis, IN) for providing UCB samples for this study, Dr. Hal Broxmeyer at Indiana University for providing human UCB CD34+ cells and helpful advice, and Mrs. Marilyn Wales for helping in the preparation of the manuscript. This work was supported in part by the Department of Defense (grant W81XWH-13-1-0187 to Y.L.), the St. Baldrick's Foundation (scholar award to Y.L.), the Elsa Pardee Foundation (Y.L.), the Leukemia Research Foundation (Y.L.), the Children's Leukemia Research Association (Y.L.), the Showalter Trust Fund (Y.L.), Alex's Lemonade Stand Foundation (research grant to Y.L.), the American Cancer Society (institutional research grant to Y.L.), and the Indiana University Simon Cancer Center (pilot project grant to Y.L.). The authors declare no competing financial interests.
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