Role of notch signaling in the maintenance of human mesenchymal stem cells under hypoxic conditions

Abstract

Human adipose tissue-derived multilineage progenitor cells (hADMPCs) are attractive for cell therapy and tissue engineering because of their multipotency and ease of isolation without serial ethical issues. However, their limited in vitro lifespan in culture systems hinders their therapeutic application. Some somatic stem cells, including hADMPCs, are known to be localized in hypoxic regions; thus, hypoxia may be beneficial for ex vivo culture of these stem cells. These cells exhibit a high level of glycolytic metabolism in the presence of high oxygen levels and further increase their glycolysis rate under hypoxia. However, the physiological role of glycolytic activation and its regulatory mechanisms are still incompletely understood. Here, we show that Notch signaling is required for glycolysis regulation under hypoxic conditions. Our results demonstrate that 5% O2 dramatically increased the glycolysis rate, improved the proliferation efficiency, prevented senescence, and maintained the multipotency of hADMPCs. Intriguingly, these effects were not mediated by hypoxia-inducible factor (HIF), but rather by the Notch signaling pathway. Five percent O2 significantly increased the level of activated Notch1 and expression of its downstream gene, HES1. Furthermore, 5% O2 markedly increased glucose consumption and lactate production of hADMPCs, which decreased back to normoxic levels on treatment with a γ-secretase inhibitor. We also found that HES1 was involved in induction of GLUT3, TPI, and PGK1 in addition to reduction of TIGAR and SCO2 expression. These results clearly suggest that Notch signaling regulates glycolysis under hypoxic conditions and, thus, likely affects the cell lifespan via glycolysis.

FIG. 1.

Hypoxia increases proliferation capacity and decreases senescence in tissue-derived multilineage progenitor cells (hADMPCs). (A) Growth profiles of hADMPCs under normoxic (red square) and hypoxic (blue square) conditions. The population doubling level (PDL) was determined to be 0 when cells were isolated from human adipose tissue. Cells were maintained until they reached PDL13–15 (passage 3) and then split into four aliquots of equal cell densities. PDL was calculated based on the total cell number at each passage. (B) Detection of normoxic (Nx) and hypoxic (Hx) cells by flow cytometry after incorporation of EdU. (C) Percentages of apoptotic cells with sub-G1 DNA under Nx and Hx conditions. The results are presented as the mean of three independent experiments. (D) hADMPCs cultured under Nx and Hx conditions were harvested by trypsin-EDTA and then imaged using a phase-contrast microscope. Arrowheads indicate cells with a larger and more irregular shape. (E) Cells expanded under Nx and Hx conditions were stained with SA-β-gal. (F) Cellular reactive oxygen species detection by the oxidative stress indicator CM-H₂DCFDA in hADMPCs under Nx or Hx. Data are presented as the mean fluorescence intensity of three independent experiments. Error bars indicate SD. **P<0.01 indicates significant difference (independent t-test) between Nx and Hx. Scale bars; 100 μm. Color images available online at www.liebertpub.com/scd

FIG. 2.

Hypoxic culture maintains mesenchymal stem cell properties. hADMPCs cultured under normoxia (20% O₂) or hypoxia (5% O₂) were labeled with antibodies against the indicated antigens and analyzed by flow cytometry. Representative histograms are shown. The respective isotype control is shown as a gray line.

FIG. 3.

Hypoxic culture enhances stem cell properties. hADMPCs were expanded under normoxic and hypoxic conditions. (A) Normoxic (20% O₂) and hypoxic (5% O₂) cells at passage 8 were induced for 3 weeks to differentiate into osteoblasts and adipocytes and stained with Alizarin Red and Oil-Red O, respectively. The stained dye was extracted, and OD values were measured and plotted as the means of three independent experiments±SD. *P<0.05. Scale bars, 200 μm. (B) Normoxic (20% O₂) and hypoxic (5% O₂) cells at passage 8 were induced for 3 weeks to differentiate to chondrocytes, and immunofluorescent analysis of collagen II (red) and Alucian Blue staining were performed. The blue signals indicate nuclear staining. Scale bars, 100 μm. Noninduced control cultures in growth medium without adipogenic, osteogenic or chondrogenic differentiation stimuli are shown (Undifferentiated). Color images available online at www.liebertpub.com/scd

FIG. 4.

Hypoxic culture condition activates Notch signaling but not HIF proteins. hADMPCs were expanded under normoxic (20% O₂) and hypoxic (5% O₂) conditions. DAPT (1 μM) was added to inhibit Notch signaling. (A) Western blot analysis of intracellular domain of Notch1 (Notch1 ICD) expression. Actin served as the loading control. Numbers below blots indicate relative band intensities as determined by ImageJ software. (B) Q-PCR analysis of HES1. Each expression value was calculated with the ΔΔCt method using UBE2D2 as an internal control. (C) Western blot analysis of HES1 in nuclear fractions of hADMPCs. Lamin A/C served as the loading control. (D, E) Western blot analysis of HIF-1α (D) and HIF-2α (E). Cobalt chloride (CoCl₂) was added at a concentration of 100 μM to stabilize HIF proteins (positive control). (F) Western blot analysis of phosphorylated Akt (p-Akt) and Akt. Actin served as the loading control. Numbers below blots indicate relative band intensities as determined by ImageJ software. (G) Western blot analysis of nuclear localization of p65. Lamin A/C served as the loading control. Numbers below blots indicate relative band intensities as determined by ImageJ software. (H) Western blot analysis of phosphorylated p53 (p-p53) and p53. Actin served as the loading control. (I) Activity of p53 was measured by the p53-luciferase reporter assay. Relative luciferase activity was determined from three independent experiments and normalized to pGL4.74 activity.

FIG. 5.

Notch signaling is indispensable for acquisition of the advantageous properties of hADMPCs. hADMPCs were expanded under normoxic (20% O₂; Nx) and hypoxic (5% O₂; Hx) conditions. DAPT (1 μM) was added to inhibit Notch signaling. (A) Growth profiles of hADMPCs under Nx (red) and Hx (blue) conditions. Solid lines represent control cells, and dotted lines represent DAPT-treated cells. The number of population doublings was calculated based on the total cell number at each passage. (B) Percentages of apoptotic cells with sub-G1 DNA. Results are presented as the mean of three independent experiments±SD. (C, D) hADMPCs at passage 8 were induced for 3 weeks to differentiate into adipocytes (C) and osteoblasts (D) and stained with Oil Red O and Alizarin Red, respectively. The stained dye was extracted, and OD values were measured and plotted as the means of three independent experiments±SD. (E) hADMPCs at passage 8 were induced for 3 weeks to differentiate into chondrocytes, and an immunofluorescent analysis of collagen II (red) was performed. The blue signals indicate nuclear staining. (F) hADMPCs were stained with SA-β-gal. *P<0.05 and **P<0.01 indicate significant differences (independent t-test) between Nx and Hx. Scale bars; 100 μm. Color images available online at www.liebertpub.com/scd

FIG. 6.

Glycolysis is enhanced under 5% oxygen through Notch signaling. (A–D) hADMPCs were expanded under normoxic (20% O₂) and hypoxic (5% O₂) conditions. DAPT (1 μM) was added to inhibit Notch signaling. (A) Glucose consumption and lactate production of hADMPCs were measured and plotted as the means of three independent experiments±SD. (B) Relative mRNA expression of SLC2A3, TPI, PGK1, TIGAR, and SCO2 in hADMPCs. Each expression value was calculated with the ΔΔCt method using UBE2D2 as an internal control. (C, D) Hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH) (C), pyruvate dehydrogenase (PDH), and Complex IV (Cox IV) (D) activities were measured and the value of relative activity was plotted as the means of three independent experiments±SD. (E, F) hADMPCs were transduced with either mock (Cont) or HES1 and then cultured for 3 days. (E) Relative mRNA expression of SLC2A3, TPI, PGK1, TIGAR, and SCO2 in hADMPCs. Each expression value was calculated with the ΔΔCt method using UBE2D2 as an internal control. (F) Glucose consumption and lactate production of hADMPCs were measured and plotted as the means of three independent experiments±SD. (G) hADMPCs were transduced with either scrambled control RNAi (Cont) or RNAi against HES1 (HES1-KD), and then cultured for 3 days. Glucose consumption and lactate production of hADMPCs were measured and plotted as the means of three independent experiments±SD. **P<0.01. *0.01<P<0.05.

FIG. 7.

Glycolysis supports proliferation of hADMPCs. hADMPCs were treated with 0, 0.2, 0.4, and 1 mM 2-deoxy-D-glucose (2-DG) (A) or 0, 1, and 5 mM sodium azide (NaN₃) (B) for 24 h. Cells were then allowed to incorporate EdU for 2 h, and the EdU-positive cells were analyzed by flow cytometry. The percentages for the 0 mM control were plotted as the means of three independent experiments±SD. *P<0.05; **P<0.01.