Hypoxic metabolism in human hematopoietic stem cells
- Fatih Kocabas†1, 2,
- Li Xie†3, 4,
- Jingjing Xie†5,
- Zhuo Yu3, 4,
- Ralph J. DeBerardinis6,
- Wataru Kimura1,
- SuWannee Thet1,
- Ahmed F. Elshamy7,
- Hesham Abouellail8,
- Shalini Muralidhar1,
- Xiaoye Liu4,
- Chiqi Chen3,
- Hesham A. Sadek1,
- Cheng Cheng Zhang9Email author and
- Junke Zheng3, 4Email author
© Kocabas et al. 2015
Received: 7 February 2015
Accepted: 5 June 2015
Published: 17 July 2015
Adult hematopoietic stem cells (HSCs) are maintained in a microenvironment, known as niche in the endosteal regions of the bone marrow. This stem cell niche with low oxygen tension requires HSCs to adopt a unique metabolic profile. We have recently demonstrated that mouse long-term hematopoietic stem cells (LT-HSCs) utilize glycolysis instead of mitochondrial oxidative phosphorylation as their main energy source. However, the metabolic phenotype of human hematopoietic progenitor and stem cells (HPSCs) remains unknown.
We show that HPSCs have a similar metabolic phenotype, as shown by high rates of glycolysis, and low rates of oxygen consumption. Fractionation of human mobilized peripheral blood cells based on their metabolic footprint shows that cells with a low mitochondrial potential are highly enriched for HPSCs. Remarkably, low MP cells had much better repopulation ability as compared to high MP cells. Moreover, similar to their murine counterparts, we show that Hif-1α is upregulated in human HPSCs, where it is transcriptionally regulated by Meis1. Finally, we show that Meis1 and its cofactors Pbx1 and HoxA9 play an important role in transcriptional activation of Hif-1α in a cooperative manner.
These findings highlight the unique metabolic properties of human HPSCs and the transcriptional network that regulates their metabolic phenotype.
KeywordsStem cells Metabolism Hypoxia Hypoxic regulation of metabolism Human hematopoietic progenitor and stem cells HPSCs
HSCs are defined by their inherent capacity for self-renewal and differentiation into all blood cell types. Adult HSCs reside in regions of the bone marrow challenged by low oxygen tension (hypoxia), which is termed “hypoxic niche” [1, 2]. We recently demonstrated that mouse HSCs in this hypoxic niche adopt a glycolytic metabolic profile, in which HSCs manifest lower rates of oxygen consumption, lower ATP content, and increased cytoplasmic glycolysis . In addition, we and others showed that mouse HSCs demonstrate increased HIF-1α levels [3–5].
HIF-1 regulates various aspects of metabolism from the oxidant stress response to regulation of glycolysis and mitochondrial respiration [6–12]. HIF-1, a major mediator of transcriptional response to hypoxia, is composed of O2 sensitive HIF-1α and constitutively active HIF-1β subunits [13–15]. Conditional deletion of HIF-1α in HSCs results in various defects such as loss of quiescence and progressive decline in HSC number following bone marrow transplantations and aging . In addition hypoxic cultures promote the production of hematopoietic progenitors and enhance HSC expansion [1, 16, 17]. HIF-1α protein is mainly stabilized during hypoxia but normoxic upregulation of HIF-1α has also been reported [14, 18–25]. We recently demonstrated that Meis1 transactivates HIF-1α expression via a conserved Meis1 binding motif located in the first intron of HIF-1α , and regulates mouse HSC metabolism .
Several reports suggest that homeobox protein Meis1 plays important roles in HPSC biology [27–32]. Homozygous mutant mice for Meis1 die during gestation with defects in hematopoiesis which results in decline in myeloid, lymphoid, and multipotent progenitors [33–35]. Meis1 is also associated with leukemogenesis in humans with a frequent up-regulation in primary acute myeloid leukemia (AML) [29, 30] and acute lymphoblastic leukemia (ALL) samples . We recently showed that mouse LT-HSCs express Meis1 where a great majority of Meis1+ HSCs coexpress Hif-1α . Previous reports indicate that Meis1 interacts with the cofactors Pbx1 and HoxA9 in HSCs, which are known regulators of hematopoiesis [27, 28, 37–48]. However, the cooperative role of Meis1 and its cofactors in regulation of HIF-1 is unknown.
In the current report, we demonstrate that human HPSCs have a metabolic profile characterized by low mitochondrial oxidative phosphorylation, dependence on glycolytic metabolism, and upregulation of hypoxia-responsive pathways. We show that the metabolic phenotype of human HPSCs is mediated in part via transcriptional activation of Hif-1α by Meis1 and its cofactors Pbx1 and HoxA9.
Metabolic profile of human HPSCs
Next we sought to profile human MPB HPSCs based on their mitochondrial proton gradient as an index of overall mitochondrial respiration. Similar to mouse HSCs, the majority of the human Lin−CD34+CD38−CD90+ cells (74 %) fell within a defined flow-cytometry gate which corresponds to 7–9 % of total G-CSF mobilized human peripheral blood cells (Fig. 1d and Additional file 1: Fig. S1a and b). Cells in this gate were characterized by low mitochondrial potential (low MP) while the majority of the remaining MPB populations fell within a high mitochondrial potential (high MP) (Fig. 1e-f). Furthermore, we determined percentages of different lineages within low and high MP cells of MPB (Fig. 1g and Additional file 1: Fig. S1c and d). The results showed the low MP cells only contained a few B cells (CD19, 7.9 %) and myeloid cells (Mac-1, 17.05 %). The high MP cells had lower percentage of B cells (CD19, 2.4 %), but much higher percentage of myeloid cells (Mac-1, 81.65 %). This result indicated that there were less differentiated cells in low MP cells compared to high MP cells, which was consistent with enrichment of HPSCs in low MP cells.
Low MP cells are primed for hypoxic niche
Increased Hif-1α expression in low MP cells suggests that low MP cells could confer survival advantage at hypoxia. Thus, we measured the viability of low MP and high MP cells at normoxia and hypoxia (Fig. 2c). Survival of low MP cells at hypoxic condition, but not at normoxic condition, was much higher than that of high MP cells. The increased survival under hypoxic conditions by low MP cells in vitro suggested that low MP cells in physiological hypoxia could have higher survival ability. To further confirm this, we examined the apoptotic status of low and high MP cells at different conditions by staining with anti-Annexin V antibodies. Consistently, we demonstrated that there was a dramatic increase of apoptosis in high MP cells at hypoxic condition, but not at normoxic condition (Additional file 2: Fig. S2a). In addition, low MP cells consumed less oxygen compared to high MP cells at normoxia while there is no significant difference at hypoxia (Fig. 2d). Moreover, measurement of glycolysis rate of low MP and high MP cells showed that the glycolytic rate is significantly increased under hypoxia, which indicated the low MP cells preferred to glycolysis as the energy source in the physiologic niche (Fig. 2e). Moreover, we used the Seahorse XF96 extracellular flux analyzer to measure the oxygen consumption rate (OCR) indicative of oxidative phosphorylation, and the lactate production indicative of glycolysis (extracellular acidification rate, ECAR). As shown in Fig. 2f and g, low MP cells showed a significantly lower OCR rate as compared to high MP cells. In contrast, low MP cells showed a significantly higher ECAR rate, indicative of higher glycolytic metabolism. This data further revealed low MP cells using glycolysis as primary energy source.
Together, these results indicate that low MP cells are characterized by baseline upregulation of glycolytic and hypoxia inducible genes, which suggests adaptation of human HPSCs to glycolysis as the major source of energy production.
Characterization of stem cell profile of metabolically sorted cells
Expression of Hif-1α and Meis1 in human HPSCs
Regulation of Hif-1α by Meis1
Cooperation of Meis1 with Pbx1 and HoxA9 in the regulation of Hif-1α expression
To further characterize the interaction of Meis1 with Pbx1 and HoxA9 proteins in the activation of Hif-1α, we generated mutant Meis1 protein lacking Pbx1 interaction Motifs (PIM) and HoxA9 protein lacking Meis1 interaction domain (MID) (Fig. 6e). As shown Additional file 6: Fig. S6, we could stably detect the expression of Meis1, Meis1∆PIM, HoxA9 and Hoxa∆MID, which indicated that deletion mutants of the proteins were stable in our system. These mutant proteins allowed us to demonstrate that protein-protein interaction between Meis1 and Pbx1 as well as Meis1 and HoxA9 are required for transcriptional activation of Hif-1α luciferase reporter by Meis1, Pbx1 and HoxA9 (Fig. 6f). In summary, Hif-1α expression is regulated by cooperative activation of Meis1, Pbx1 and HoxA9 (Fig. 6g). This activation is dependent on consensus binding motifs located at Hif-1α intronic region.
Several studies indicated that human HPSCs reside in hypoxic niches and are capable of surviving upon hypoxic stress [1, 16, 17]. However, it is unclear if HPSCs are endowed with distinct metabolic properties that confer this hypoxic tolerance. In the current report, we show that human HPSCs are localized to a flow cytometry gate characterized by low mitochondrial potential (low MP). This population represents only 7–9 % of the G-CSF mobilized human peripheral blood cells, but is enriched for more than 70 % of human HPSCs as Lin−CD34+CD38−CD90+ cells. Moreover, we confirm this metabolic profile by demonstrating that human HPSCs have lower ATP content, lower rates of oxygen consumption, and use cytoplasmic glycolysis instead of mitochondrial oxidative phosphorylation. Interestingly, here we demonstrated that the glycolytic phenotype of HPSCs persists in circulating cells, suggesting that the metabolic phenotype of HSCs is a product of intrinsic regulatory mechanisms and not just their environment. Similar to their murine counterparts, this unique metabolic profile is associated with upregulation of Hif-1α. It is noteworthy that we found human HPSCs are localized to the low MP gate even after G-CSF mobilization to the peripheral blood, which indicates that HPSCs may have intrinsic mechanism for the regulation of their metabolic properties, other than their location in hypoxic niches.
Hif-1α as a master regulator of metabolism plays an essential role in various aspects of adaptation to hypoxic stress. Regulation of Hif-1α expression is well studied in terms of its posttranslational stabilization during hypoxia, however, little is known about its transcriptional regulation. Previous reports by our group and others indicate that Hif-1α is regulated at the transcription level not only at hypoxia but also normoxia [3, 22, 49–51]. Here we show that, the transcription factor Meis1 plays an important role in HPSC metabolism through transcriptional regulation of Hif-1α in cooperation with its cofactors Pbx1 and HoxA9. Meanwhile, we also noticed that anyone of Meis1, Pbx1 or HoxA9 was able to upregulate Hif-1α at transcription level (Fig. 6d). However, when one or more mutated factor(s) plus another WT factor did not transcriptionally upregulate Hif-1α (Fig. 6f). We speculated that the expression of mutant forms of the any protein might interrupt the action of any WT protein in transcriptional regulation of Hif-1α. This might indicate that normal expression levels of these factors play an important role in the synergistic effect in the expression of Hif-1α. Recently, our group recently demonstrated that Meis1 is an important regulator of postnatal cardiomyocyte cell cycle . Intriguingly, we found that Pbx1 and HoxA9 were not expressed in postnatal cardiomyocytes, while other AbdB-like paralogue of Hox genes seem to have an expression pattern similar to Meis1. Therefore, it may be that the transcriptional activity of Meis1 in various compartments is regulated through differential expression pattern of its cofactors.
In summary, our findings demonstrate the unique metabolic properties of human HPSCs as compared to human MPB mononuclear cells, as well as the transcriptional network that regulates their metabolic phenotype. The transcriptional regulation of Hif-1α by Meis1 and its cofactors, and the metabolic shift that follows loss of Meis1 suggest that the metabolic profile of human HPSCs is an intrinsic characteristic of these cells and not only a product of their hypoxic microenvironment. It is therefore important for future studies to determine the role of these unique metabolic properties in vitro human HPSC culture and expansion, as well as in designing therapeutic strategies for hematopoietic disorders.
Isolation of human mobilized peripheral blood (MPB) cells
Human G-CSF mobilized peripheral blood (MPB) was obtained from bone marrow transplant donors by plasmapheresis. Human cells were diluted and separated using ficoll to obtain the MPB mononuclear cell fraction. Then cells were suspended at 2–3 × 106 cells/mL in DMEM media with 10 % fetal bovine serum for flow cytometry. The study was approved by the Institutional Review Board of affiliated hospital of UT Southwestern Medical Center (Dallas, USA) and Shanghai JiaoTong University School of Medicine (Shanghai, China). Written informed consent was obtained from each donor for his enrollment. All clinical investigation was conducted in accordance with the principles expressed in the Declaration of Helsinki.
Flow cytometric profiling and separation of cells based on mitochondrial potential
Flow cytometric profiling and separation of human MPB cells based on their mitochondrial activity were performed by using mitotracker dyes as described previously . Briefly, human MPB cells were enriched by lineage depletion using the BD IMag™ Human Lineage Cell Depletion Set (BD Biosciences) according to manufacturer’s instructions. The whole MPB fraction and Lin−CD34+CD38−CD90+ cells (30 min incubation on ice) were then profiled based on their mitochondrial activity after 15 min staining with mitotracker dyes (MitoTracker Red FM Cat# M22425 and MitoTracker Green Cat# M7514, Invitrogen) at 37 °C (2 × 106 cells/mL, 200 nM mitotracker). Cell sorting based on high and low mitochondrial activity was performed using gates that separated the cells with those with the lowest 7–9 % of mitochondrial activity and equivalent number of cells with the high mitochondrial activity (named low and high mitochondrial potential (MP) cells).
Colony forming assay and in vivo transplantation
Based on mitochondrial profile, colony forming cell (CFC) assays were performed on human G-CSF MPB cells. Same numbers of human high and low MP cells (3 × 104 cells) were plated in each methocult plate (MethoCult® GF H4434, Stem Cell Technologies, USA). Viability was assessed with trypan blue as this is crucial that low MP gate often have dying cells with low mitochondrial activity. To minimize cell attachment, plates were precoated with 1 % agarose. After twelve days of culture, the total number of colonies (per plate) was counted and the types of colonies were quantified. Note CFU-GEMM colonies indicate mixed colonies whereas BFU-E and CFU-GM are measure of erythroid and myeloid progenitors, respectively. For in vivo transplantation, NOD-SCID mice were purchased from the Shanghai SLAC Laboratory Animal Co. Ltd. Five millions of FACS-sorted low MP or high MP cells in 50 μL of PBS were directly injected into the tibia as described previously . Human engrafted cells in bone marrow were detected by using anti-human CD45 antibodies 2 months after transplantation.
Oxygen consumption was determined using the BD Oxygen Biosensor System, 384 well (Cat#353,834) according to manufacturer’s recommendations. MPB Cells and human HPSCs were separated as described above. Equal numbers of cells (5 × 104 cells/well in 50 μL volume) were incubated up to 6 h in the provided 384 well plate prior to measurement at Fluostar Optima plate reader (BMG Labtech). Culture media lacking cells was used as a negative control and sodium sulfite (100 mM) was used as a positive control. Oxygen consumption is presented as relative units.
HPSCs from human MPB Cells were isolated as described. Fifty thousand cells were used for each measurement. HPSCs and MPB Cells were centrifuged at 1200 g for 10 min. ATP standard curves were prepared using ATP concentrations between 10−6-10−12 M. Then, 50 μl of ATP standards and 50 μl cell lysates were quantified using ATP Bioluminescence Assay Kit HS II (Roche) using Fluostar Optima plate reader (BMG Labtech) following manufacturer instructions. Finally, data were normalized to cell count.
Measurement of 13C lactate production
Cells were cultured in DMEM (Sigma D5030) supplemented with L-glutamine (4 mM), NaHCO3 (42.5 mM), HEPES (25 mM), dialyzed fetal calf serum (10 % v/v), Penicillin/Streptomycin and no glucose per well of 96-well plate. The medium was supplemented with double labeled 10 mM D-[1-13C, 6-13C]-glucose (Cambridge Isotope Labs) to allow all of the glucose-derived lactate pool to be labeled on C-3. Fifty thousand cells (each well) were cultured in 40 μL of medium for 12 h. After the culture, the cells were pelleted and an aliquot of 25 μL of the medium was transferred to a glass test tube with the internal standard (17.9 nmol of Sodium L-[13C3]-Lactate, Cambridge Isotope Labs). Lactate was extracted by sequential addition of 1 mL of methanol, chloroform and water followed by vortexing and centrifugation at 2000 rpm for 5 min. The aqueous phase was evaporated, derivatized with 100 μL Tri-Sil reagent (Pierce) for 30 min at 42 °C, and analyzed by gas chromatography–mass spectrometry. A three-point standard curve was also prepared using mixtures of un-enriched lactate and L-[3-13C3]-lactate (Cambridge Isotope Labs). Lactate abundance was determined by monitoring m/z at 117 (un-enriched), 118 (lactate containing 13C from glucose) and 119 (internal standard). The areas of 117 and 118 were summed and corrected against the 119 area to calculate total lactate abundance. To determine the atom percent excess (APE), the 117 and 118 areas were first corrected against the 119 abundance to account for inter-sample variability of extraction. Then the corrected ratio of 118/(117 + 118) was determined and compared to the standard curve. Finally, the APE was multiplied by the total nmoles lactate to determine the nmoles of 13C-lactate produced. The final results were corrected for total cellular ATP concentration.
Metabolic assays with seahore XF96
Oxygen consumption and lactate generation were measured using the Seahorse XF96 extracellular flux analyzer as previously described . Briefly, three replicate wells of 3 × 105 low MP or high MP cells per well were seeded overnight in 96-well XF96 well plates coated with BD Cell-Tak (BD Biosciences) in serum-free culture medium containing 20 ng/ml SCF and 20 ng/ml TPO. One hour prior to analysis, the medium was replaced by unbuffered DMEM and the well plates were incubated to 37°C for pH stabilization. Analyses were performed both at basal conditions and after injection of oligomycin (0.5 μM for OCR or 2 μM for ECAR), FCCP (2 μM), Antimycin A (0.5 μM), Glucose (10 mM), and 2-DG (100 mM).
Meis1 siRNA knockdown was carried out in Kasumi-1 cells (human myeloid progenitor cell line known to express Hif-1α at normoxic conditions ). Upregulation of Meis1 in low MP cells and Kasumi-1 cells compared to human MPB cells were confirmed by real time PCR. Kasumi-1 cells were diluted to a density of 200,000 cells per mL in RPMI media supplemented with 20 % FBS and antibiotics. The cells were centrifuged for 10 min at 1200 g at 4 °C and suspended in OPTIMEM (Invitrogen). The cells were then plated into 6-well plates. 12 μL of Hiperfect (Qiagen) and siRNA (50nM of siRNA per 750,000 cells/well) were incubated in 200 μL of OPTIMEM (Invitrogen) for 20 min at RT. Silencer Select Pre-designed siRNAs (Applied Biosystems, Ambion) for Meis1 were diluted into 50nM stocks (siRNA ID# s8662: 5’-GGCAUCUACUCGUUCAGGAtt-3’ and 5’-UCCUGAACGAGUAGAUGCCgt-3’). siRNA were added to cells dropwise and incubated for 6 h. After 6 h incubation, 2.5 mL RPMI medium was added (20 % FBS and antibiotics) and plates were incubated under normal growth conditions for 48 h.
Expression profile of low and high MP cells
PCR arrays (HSC and hypoxia primer sets, SABiosciences) were performed to study HPSC and hypoxia inducible gene expression profile of low and high MP cells. RNA was isolated from low and high MP cells by TRIzol method (Invitrogen, Carlsbad, CA) following manufacturer instructions. cDNA was generated by using an RT-PCR kit (SA biosciences, Maryland, USA). Real-Time PCR was performed using Human Real-Time Syber Green PCR Mix (SuperArray) on an ABI Prism 7700 Sequence Detector (Applied Biosystems). The data was analyzed using the ΔΔCt method. Fold change was calculated as difference in gene expression between low and high MP cells.
Real time PCR for Meis1, Hif-1α and Hif-1α related genes
Primers used in this study
Gene [GenBank: Accession Numbers]
Intracellular detection of Hif-1α and Meis1 in HPSCs
Human MPB cells and human HPSCs underwent fixation with 4 % paraformaldehyde for 10 min at room temperature. After permeabilization (0.01 % Triton) and serum block, cells were incubated overnight with primary antibodies; 1:50 dilution of anti-Hif-1α (Cat#610958, BD Transcduction Laboratories) and 1:50 dilution of anti-Meis1 (sc-10599, Santa Cruz Biotechnology). Staining was assessed by flow cytometry after incubation with corresponding fluorophore-conjugated secondary antibody.
Generation of Meis1 lacking Pbx1 interaction motifs and HoxA9 lacking Meis1 interaction domain constructs
Meis1 and Pbx1 protein interaction were assessed by deletion of Pbx1 interaction domain of Meis1. For that purpose, we generated Meis1 lacking aminoacids for Pbx1 interaction domain (both 71–100 aa HR1 and 136-180aa HR regions) by polymerase chain reaction (PCR) from pCMV-SPORT6-Meis1 (MMM1013-7512739, Openbiosystems) using primers (NotI site were inserted for screening) Meis1dPIM(71–130)F: 5’- ATCTATcatgcggccgcAAAATGCCTATCGATTTGGTGATAG-3’ and Meis1dPIM(71–130)R: 5’-ATCTATcatgcggccgcTAAAGCGTCATTGACCGAGGAACCC-3’.
Meis1 and HoxA9 protein interaction were studied by co-tranfection of Meis1 with HoxA9 lacking Meis1 interaction domain (MID). HoxA9 lacking aminoacids for Meis1 interaction domain (1–61) were generated by PCR from pCMV-SPORT6-HoxA9 (MMM1013-9200573, Openbiosystems) using primers (BamHI site were inserted for screening) HoxA9dN(1–61)F: 5’-ATCTATcatggatccCATTGCAGTAGCCCGCGCCTG GCCGG-3’ and HoxA9dN(1–61)R: 5’-ATCTATcatggatccGCGGTGTTTGGTGCCTCGTGGAACCC-3’.
Transcriptional activation of Hif-1α reporter by Meis1 (pCMV-SPORT6-Meis1, Cat#MMM1013-7512739,Openbiosystems), Meis1 lacking Pbx1 interaction domain (Meis1∆PIM), HoxA9 (pCMV-SPORT6-HoxA9, Cat# MMM1013-9200573, Openbiosystems), HoxA9 lacking Meis1 interaction domain (Hoxa∆MID) and Pbx1 (pCMV-SPORT6-Pbx1, Cat# MMM1013-63227,Openbiosystems), were evaluated using Hif-1α-pGL2 or PbxMut-Hif-1α-pGL2 (having Pbx1 binding site mutated) or HoxMut-Hif-1α-pGL2 (having HoxA9 binding site mutated) reporter constructs as indicated in the figures. 0.8 μg of Hif-1α-pGL2 or PbxMut-Hif-1α-pGL2 or HoxMut-Hif-1α-pGL2 reporter construct were co-transfectedwith different doses of the Meis1, Meis1∆PIM, HoxA9, Hoxa∆MID and Pbx1 expression vectors and 0.2 μg of pCMV-LacZ (internal control) into COS cells using lipofectamine transfection reagent (Invitrogen). At 48 h after transfection, cell lysate were prepared and quantified for firefly luciferase activity using a luciferase reporter system (Promega). Luciferase measurements were calculated as firefly luciferase units versus b-gal units. Transcriptional activation was compared to basal luciferase levels in cells transfected with Hif-1α-pGL2 or PbxMut-Hif-1α-pGL2 or HoxMut-Hif-1α-pGL2 and empty pGL2. To test Pbx1 site specificity, Pbx1 binding site TGAT at Hif-1α-pGL2 reporter were mutated using site directed mutagenesis kit (Strategene) from Hif-1α-pGL2 vector with the following primers: PbxMut-Hif-1α-F:5’-CAATTTCTACAAACTTGTGTTTGCCG ACCCTGTCAGGAGAGCCCAGACGTTA-3’ and PbxMut-Hif-1α-R: 5’-TAACGTCTGGGCTCTCCTGACAGGGTC GGCAAACACAAGTTTGTAGAAATTG-3’. Similarly, to test HoxA9 site specificity, HoxA9 binding site ATAA at Hif-1α reporter using site directed mutagenesis kit from Hif-1α-pGL2 vector with the following primers: HoxMu-Hif-1α-F: 5’-CCTGTCAGGAGAGCCCAGACGCCGCGTATCAATATGTGGCTG CCTC-3’ and HoxMut-Hif-1α-R: 5’-GAGGCAGCCACATATTGATACGCGGCGTCTG GGCTCTCCTGACAGG-3’.
Western blotting and apoptosis assay
Plasmids of pCMV-SPORT6-Meis1, Meis1∆PIM, pCMV-SPORT6-HoxA9 and Hoxa∆MID were transfected into 293 T cells. Whole cell lysates were electrophoresed on 8–10 % sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were incubated with primary antibodies overnight at 4 °C followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies. The following antibodies were used: anti-Meis1 (Abcam), anti-HoxA9 (Proteintech) and anti-β-actin (Calbiochem). For analysis of apoptosis, cells were stained with PE conjugated anti-annexin V and 7-AAD (BD Pharmingen) according to the manufacturer’s instructions.
Results are expressed as mean ± SEM and a 2-tailed Student t test was used to determine the level of significance. p < 0.05 was considered statistically different.
Hematopoietic stem cells
Human hematopoietic progenitor and stem cells
Hypoxia-inducible factor 1-alpha
Meis homeobox 1
Pre-B-cell leukemia transcription factor 1
This work is supported by grants from the American Heart Association (AHA Fellow to Faculty Award) (Sadek), the Gilead Research Scholars Program in Cardiovascular Disease (Sadek), NIH grant 1R01CA172268 (Zhang), CPRIT RP140402 (Zhang), March of Dimes Foundation Award #1-FY14-201 (Zhang), National Natural Science Foundation of China (81370654, 81471524 and 31171409), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning and the Pujiang Program (13PJ1405600) (Zheng). We thank the support from CO-FUNDED Brain Circulation Scheme by The Scientific and Technological Research Council of Turkey (TÜBİTAK) and The Marie Curie Action COFUND of the 7th. Framework Programme (FP7) of the European Commission grant 115C039 (Kocabas), The Science Academy of Turkey Young Scientist Award Program (Kocabas), and funds provided by Yeditepe University, Istanbul, Turkey (Kocabas).
- Eliasson P, Jonsson JI. The hematopoietic stem cell niche: low in oxygen but a nice place to be. J Cell Physiol. 2010;222(1):17–22.PubMedView ArticleGoogle Scholar
- Wang LD, Wagers AJ. Dynamic niches in the origination and differentiation of haematopoietic stem cells. Nat Rev Mol Cell Biol. 2011;12(10):643–55.PubMed CentralPubMedView ArticleGoogle Scholar
- Simsek T, Kocabas F, Zheng JK, DeBerardinis RJ, Mahmoud AI, Olson EN, et al. The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche. Cell Stem Cell. 2010;7(3):380–90.PubMed CentralPubMedView ArticleGoogle Scholar
- Takubo K, Goda N, Yamada W, Iriuchishima H, Ikeda E, Kubota Y, et al. Regulation of the HIF-1alpha level is essential for hematopoietic stem cells. Cell Stem Cell. 2010;7(3):391–402.PubMedView ArticleGoogle Scholar
- Kim CG, Lee JJ, Jung DY, Jeon J, Heo HS, Kang HC, et al. Profiling of differentially expressed genes in human stem cells by cDNA microarray. Mol Cells. 2006;21(3):343–55.PubMedGoogle Scholar
- Papandreou I, Cairns RA, Fontana L, Lim AL, Denko NC. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab. 2006;3(3):187–97.PubMedView ArticleGoogle Scholar
- Kim JW, Tchernyshyov I, Semenza GL, Dang CV. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab. 2006;3(3):177–85.PubMedView ArticleGoogle Scholar
- Marin-Hernandez A, Gallardo-Perez JC, Ralph SJ, Rodriguez-Enriquez S, Moreno-Sanchez R. HIF-1alpha modulates energy metabolism in cancer cells by inducing over-expression of specific glycolytic isoforms. Mini Rev Med Chem. 2009;9(9):1084–101.PubMedView ArticleGoogle Scholar
- Zhang H, Gao P, Fukuda R, Kumar G, Krishnamachary B, Zeller KI, et al. HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deficient renal cell carcinoma by repression of C-MYC activity. Cancer Cell. 2007;11(5):407–20.PubMedView ArticleGoogle Scholar
- Semenza GL. Regulation of cancer cell metabolism by hypoxia-inducible factor 1. Semin Cancer Biol. 2009;19(1):12–6.PubMedView ArticleGoogle Scholar
- Semenza GL. Regulation of oxygen homeostasis by hypoxia-inducible factor 1. Physiology (Bethesda). 2009;24:97–106.View ArticleGoogle Scholar
- Semenza GL. Oxygen homeostasis. Wiley Interdiscip Rev Syst Biol Med. 2010;2(3):336–61.PubMedView ArticleGoogle Scholar
- Semenza GL. HIF-1 and mechanisms of hypoxia sensing. Curr Opin Cell Biol. 2001;13(2):167–71.PubMedView ArticleGoogle Scholar
- Semenza GL. Hypoxia-inducible factor 1 (HIF-1) pathway. Sci STKE. 2007;2007(407):cm8.PubMedView ArticleGoogle Scholar
- Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A. 1995;92(12):5510–4.PubMed CentralPubMedView ArticleGoogle Scholar
- Parmar K, Mauch P, Vergilio JA, Sackstein R, Down JD. Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia. Proc Natl Acad Sci U S A. 2007;104(13):5431–6.PubMed CentralPubMedView ArticleGoogle Scholar
- Koller MR, Bender JG, Miller WM, Papoutsakis ET. Reduced oxygen tension increases hematopoiesis in long-term culture of human stem and progenitor cells from cord blood and bone marrow. Exp Hematol. 1992;20(2):264–70.PubMedGoogle Scholar
- Salceda S, Caro J. Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by theubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia dependson redox-induced changes. J Biol Chem. 1997;272(36):22642–7.PubMedView ArticleGoogle Scholar
- Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, et al. HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science. 2001;292(5516):464–8.PubMedView ArticleGoogle Scholar
- Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 2001;292(5516):468–72.PubMedView ArticleGoogle Scholar
- Qi J, Nakayama K, Gaitonde S, Goydos JS, Krajewski S, Eroshkin A, et al. The ubiquitin ligase Siah2 regulates tumorigenesis and metastasis by HIF-dependent and -independent pathways. Proc Natl Acad Sci U S A. 2008;105(43):16713–8.PubMed CentralPubMedView ArticleGoogle Scholar
- BelAiba RS, Bonello S, Zharinger C, Schmidt S, Hess J, Kietzmann T, et al. Transcriptional regulation of HIF-1alpha by NFkappaB in response to hypoxia. Circulation. 2006;114(18):182–3.Google Scholar
- Pedersen M, Lofstedt T, Sun J, Holmquist-Mengelbier L, Pahlman S, Ronnstrand L. Stem cell factor induces HIF-1alpha at normoxia in hematopoietic cells. Biochem Biophys Res Commun. 2008;377(1):98–103.PubMedView ArticleGoogle Scholar
- Belaiba RS, Bonello S, Zahringer C, Schmidt S, Hess J, Kietzmann T, et al. Hypoxia up-regulates hypoxia-inducible factor-1alpha transcription by involving phosphatidylinositol 3-kinase and nuclear factor kappaB in pulmonary artery smooth muscle cells. Mol Biol Cell. 2007;18(12):4691–7.PubMed CentralPubMedView ArticleGoogle Scholar
- Hagg M, Wennstrom S. Activation of hypoxia-induced transcription in normoxia. Exp Cell Res. 2005;306(1):180–91.PubMedView ArticleGoogle Scholar
- Kocabas F, Zheng J, Thet S, Copeland NG, Jenkins NA, DeBerardinis RJ, et al. Meis1 regulates the metabolic phenotype and oxidant defense of hematopoietic stem cells. Blood. 2012;120(25):4963–72.PubMed CentralPubMedView ArticleGoogle Scholar
- Pineault N, Helgason CD, Lawrence HJ, Humphries RK. Differential expression of Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic ontogeny. Exp Hematol. 2002;30(1):49–57.PubMedView ArticleGoogle Scholar
- Argiropoulos B, Yung E, Humphries RK. Unraveling the crucial roles of Meis1 in leukemogenesis and normal hematopoiesis. Genes Dev. 2007;21(22):2845–9.PubMedView ArticleGoogle Scholar
- Eppert K, Takenaka K, Lechman ER, Waldron L, Nilsson B, van Galen P, et al. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med. 2011;17(9):1086–93.PubMedView ArticleGoogle Scholar
- Becker MW, Jordan CT. Leukemia Stemness Signatures Step toward the Clinic. Cell Stem Cell. 2011;9(3):185–6.PubMedView ArticleGoogle Scholar
- Wang GG, Pasillas MP, Kamps MP. Meis1 programs transcription of FLT3 and cancer stem cell character, using a mechanism that requires interaction with Pbx and a novel function of the Meis1 C-terminus. Blood. 2005;106(1):254–64.PubMed CentralPubMedView ArticleGoogle Scholar
- Wang GG, Pasillas MP, Kamps MP. Persistent transactivation by meis1 replaces hox function in myeloid leukemogenesis models: evidence for co-occupancy of meis1-pbx and hox-pbx complexes on promoters of leukemia-associated genes. Mol Cell Biol. 2006;26(10):3902–16.PubMed CentralPubMedView ArticleGoogle Scholar
- Hisa T, Spence SE, Rachel RA, Fujita M, Nakamura T, Ward JM, et al. Hematopoietic, angiogenic and eye defects in Meis1 mutant animals. EMBO J. 2004;23(2):450–9.PubMed CentralPubMedView ArticleGoogle Scholar
- Azcoitia V, Aracil M, Martinez-A C, Torres M. The homeodomain protein Meis1 is essential for definitive hematopoiesis and vascular patterning in the mouse embryo. Dev Biol. 2005;280(2):307–20.PubMedView ArticleGoogle Scholar
- Imamura T, Morimoto A, Takanashi M, Hibi S, Sugimoto T, Ishii E, et al. Frequent co-expression of HoxA9 and Meis1 genes in infant acute lymphoblastic leukaemia with MLL rearrangement. Br J Haematol. 2002;119(1):119–21.PubMedView ArticleGoogle Scholar
- Kawagoe H, Humphries RK, Blair A, Sutherland HJ, Hogge DE. Expression of HOX genes, HOX cofactors, and MLL in phenotypically and functionally defined subpopulations of leukemic and normal human hematopoietic cells. Leukemia. 1999;13(5):687–98.PubMedView ArticleGoogle Scholar
- Huang Y, Sitwala K, Bronstein J, Sanders D, Dandekar M, Collins C, et al. Identification and characterization of Hoxa9 binding sites in hematopoietic cells. Blood. 2011;119(2):388–98.PubMedView ArticleGoogle Scholar
- Ficara F, Murphy MJ, Lin M, Cleary ML. Pbx1 regulates self-renewal of long-term hematopoietic stem cells by maintaining their quiescence. Cell Stem Cell. 2008;2(5):484–96.PubMed CentralPubMedView ArticleGoogle Scholar
- Calvo KR, Knoepfler P, McGrath S, Kamps MP. An inhibitory switch derepressed by pbx, hox, and Meis/Prep1 partners regulates DNA-binding by pbx1 and E2a-pbx1 and is dispensable for myeloid immortalization by E2a-pbx1. Oncogene. 1999;18(56):8033–43.PubMedView ArticleGoogle Scholar
- Hu YL, Fong S, Ferrell C, Largman C, Shen WF. HOXA9 modulates its oncogenic partner Meis1 to influence normal hematopoiesis. Mol Cell Biol. 2009;29(18):5181–92.PubMed CentralPubMedView ArticleGoogle Scholar
- Cvejic A, Serbanovic-Canic J, Stemple DL, Ouwehand WH. The role of meis1 in primitive and definitive hematopoiesis during zebrafish development. Haematologica. 2011;96(2):190–8.PubMed CentralPubMedView ArticleGoogle Scholar
- Pillay LM, Forrester AM, Erickson T, Berman JN, Waskiewicz AJ. The Hox cofactors Meis1 and Pbx act upstream of gata1 to regulate primitive hematopoiesis. Dev Biol. 2010;340(2):306–17.PubMedView ArticleGoogle Scholar
- Ferrell CM, Dorsam ST, Ohta H, Humphries RK, Derynck MK, Haqq C, et al. Activation of stem-cell specific genes by HOXA9 and HOXA10 homeodomain proteins in CD34+ human cord blood cells. Stem Cells. 2005;23(5):644–55.PubMedView ArticleGoogle Scholar
- Gwin K, Frank E, Bossou A, Medina KL. Hoxa9 regulates Flt3 in lymphohematopoietic progenitors. J Immunol. 2010;185(11):6572–83.PubMed CentralPubMedView ArticleGoogle Scholar
- Lawrence HJ, Christensen J, Fong S, Hu YL, Weissman I, Sauvageau G, et al. Loss of expression of the Hoxa-9 homeobox gene impairs the proliferation and repopulating ability of hematopoietic stem cells. Blood. 2005;106(12):3988–94.PubMed CentralPubMedView ArticleGoogle Scholar
- Thorsteinsdottir U, Mamo A, Kroon E, Jerome L, Bijl J, Lawrence HJ, et al. Overexpression of the myeloid leukemia-associated Hoxa9 gene in bone marrow cells induces stem cell expansion. Blood. 2002;99(1):121–9.PubMedView ArticleGoogle Scholar
- Yan J, Chen YX, Desmond A, Silva A, Yang Y, Wang H, et al. Cdx4 and menin co-regulate Hoxa9 expression in hematopoietic cells. PLoS One. 2006;1, e47.PubMed CentralPubMedView ArticleGoogle Scholar
- Kirito K, Fox N, Kaushansky K. Thrombopoietin induces HOXA9 nuclear transport in immature hematopoietic cells: potential mechanism by which the hormone favorably affects hematopoietic stem cells. Mol Cell Biol. 2004;24(15):6751–62.PubMed CentralPubMedView ArticleGoogle Scholar
- Laughner E, Taghavi P, Chiles K, Mahon PC, Semenza GL. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1 alpha (HIF-1 alpha) synthesis: Novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol. 2001;21(12):3995–4004.PubMed CentralPubMedView ArticleGoogle Scholar
- Suda T, Takubo K, Semenza GL. Metabolic regulation of hematopoietic stem cells in the hypoxic niche. Cell Stem Cell. 2011;9(4):298–310.PubMedView ArticleGoogle Scholar
- Zhang CC, Sadek HA. Hypoxia and metabolic properties of hematopoietic stem cells. Antioxid Redox Signal. 2014;20(12):1891–901.PubMed CentralPubMedView ArticleGoogle Scholar
- Mahmoud AI, Kocabas F, Muralidhar SA, Kimura W, Koura AS, Thet S, et al. Meis1 regulates postnatal cardiomyocyte cell cycle arrest. Nature. 2013;497(7448):249–53.PubMed CentralPubMedView ArticleGoogle Scholar
- Anderson K, Lutz C, van Delft FW, Bateman CM, Guo Y, Colman SM, et al. Genetic variegation of clonal architecture and propagating cells in leukaemia. Nature. 2011;469(7330):356–61.PubMedView ArticleGoogle Scholar
- Varum S, Rodrigues AS, Moura MB, Momcilovic O, Easley CA, Ramalho-Santos J, et al. Energy metabolism in human pluripotent stem cells and their differentiated counterparts. PLoS ONE. 2011;6(6):e20914.PubMed CentralPubMedView ArticleGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.