Hypoxic Preconditioning of Mesenchymal Stromal Cells Induces Metabolic Changes, Enhances Survival, and Promotes Cell Retention In Vivo

Cell Proliferation and Cell Cycle Analysis

For proliferation assays, MSCs were seeded into 12-well-plates at 1,000 cells per square centimeter. At every time point (2–3 days), cells were lifted with trypsin treatment and counted using Trypan blue exclusion dye (#15250–061, Gibco) and a hemocytometer.

To determine the status in cell cycle, MSCs were seeded into 75 cm² culture flasks at 500 cells per square centimeter and incubated in standard culture medium with varying levels of hypoxia for 4 days, with media change at day 2. Then, cells were lifted by trypsin treatment and permeabilized with ice-cold 70% ethanol overnight. After 45 minutes incubation with a 50 μg/ml propidium iodide solution (#13301000, Roche) containing 20 ng/ml RNase A (#19101, Qiagen), followed by a washing step with PBS, samples were inspected by flow cytometry and analyzed using ModFit LT (Verity Software House).

Osteogenic and Adipogenic Differentiation Assays

For both osteogenesis and adipogenesis, 10,000 MSCs/cm² were cultured for 14 days in incubators with varying hypoxia levels (20%, 10%, 5%, and 1% O₂), with media changes every 3 days. Osteogenic medium consisted of standard culture medium supplemented with 0.2 mM ascorbic acid, 0.1 μM dexamethasone, and 20 mM β-glycerolphosphate. Adipogenic medium was standard culture medium with 0.5 mM isobutylmethylxanthine, 50 μM indomethacin, and 0.5 μM dexamethasone.

To quantify osteogenesis, alkaline phosphatase (ALP) activity was measured by lifting the cells with trypsin and lysing the cells for protein extraction using 1.5 mM Tris-HCl solution containing 1.0 mM ZnCl₂, 1.0 mM MgCl₂, and 1% Triton X-100 for 15 minutes. Lysates were then centrifuged at 12,000g for 10 minutes and incubated with p-nitrophenylphosphate liquid substrate solution (#3368-04-5, Sigma-Aldrich) for 30 minutes. Released p-nitrophenolate was determined spectrophotometrically at 405 nm. Calcium precipitation was determined using Alizarin Red S indicator (ARS; #500–4, Ricca Chemicals). Cells were fixed with 10% vol/vol formalin solution for 15 minutes, washed once with PBS, and stained for 20 minutes with 1% wt/vol ARS over gentle shaking. Samples were then photographed for visual documentation and then incubated with 10% vol/vol acetic acid for 30 minutes, the cell layer scraped, vortexed for 30 seconds, then centrifuged at 12,000g for 10 minutes. Optic density of the supernatants was measured at 405 nm. For both, ALP and ARS, total protein concentration was determined with Coomassie staining and measured at 595 nm. To determine expression of osteogenic markers, total RNA was extracted using a Direct-zol RNA MiniPrep kit (#R2050, Zymo Research). Reverse transcription was performed using Taqman Reverse Transcription Reagents (#N808–0234, Life Technologies), mRNA levels measured by real-time PCR using SYBRGreen (#4385612, Life Technologies), and primers indicated in the legend of Supporting Information Figure S3.

For adipogenesis, cells were fixed with 10% vol/vol formalin for 15 minutes, rinsed once with PBS, and stained for 30 minutes with oil red O (#26609–01, Electron Microscopy Sciences). Cells were then washed three times with PBS, photographed, and incubated with 4% Tween 20 (#9005-64-5, Affymetrix) in isopropanol for 5 minutes, in order to release the dye. Optic density of supernatants was then measured at 490 nm. To measure expression of adipogenic markers, total RNA and reverse transcription were performed as described above. Real-time PCR was done using Applied Biosystems taq-man, using the sets shown in the respective figure legend.

In Vitro Cell Survival and Apoptosis Assays

To determine survival of MSCs in hypoxia and serum deprivation we first incubated MSCs (10,000 cells per square centimeter) in 12-well plates in standard culture medium and 1% O₂, for varying times (0, 16, 48, or 96 hours). Then, media were changed to MEMa alone (without FBS) and all plates were transferred to 1% O₂ for up to 12 days with no additional media changes. Every 3 days, cells were detached by trypsin treatment and counted using Trypan blue exclusion dye and a hemocytometer.

To quantify the percentage of dead cells, MSCs were cultured on glass coverslips (10,000 cells per square centimeter) and incubated for 48 hours in standard culture medium in 20% O₂ (control) or 1% O₂ (HP). Then, medium was changed to serum-free medium and plates were transferred to 1% O₂ for 9 days with no additional media changes. Coverslips were then incubated in 4 μM Ethidium homodimer III (EthD-III; #99905, Biotium) in PBS for 30 minutes and then mounted on slides with Vectashield Mounting Medium with DAPI (#H-1200, Vector Laboratories). Images were acquired and processed for large-field merge using a BZ-9000 (BIOREVO) fluorescence microscope (Keyence) and analyzed for automated counting with NIS-Elements BR software (Nikon).

To quantify the percentage of apoptotic cells, MSCs were cultured as described above for EthD-III, but in six-well plates. At day 9, cells were lifted using trypsin and stained with PE Annexin V Apoptosis Detection Kit I (#559763, BD Pharmin-gen) following manufacturer’s instructions and measured by flow cytometry.

Apoptosis Array and Western Blot

To determine differences in apoptotic pathways, samples were prepared as follows: MSCs were incubated in standard culture medium in 20% or 1% O₂ for 48 hours, then medium was changed to MEMa alone (without FBS) and all plates transferred to 1% O₂. After 3 days, cells were lysed for protein extraction using RIPA Buffer (#89901, Thermo Scientific) containing 1% Halt Proteinase and Phosphatase Inhibitor Cocktail (#1861281, Thermo Scientific). Proteins were further extracted by strong agitation for 20 minutes on ice, then centrifuged at 12,000g for 10 minutes and stored at −80°C. Protein extracts were then analyzed using a Human Apoptosis Antibody Array (#ARY009, R&D Systems) following manufacturer’s instructions.

For Western blots, MSC culture and protein extracts were performed as described for the Apoptosis Proteome Array. Protein extracts (30 μg) were loaded into polyacrylamide gels (#456–8094, BioRad) and transferred into polyvinylidene fluoride membranes (#162–0174, BioRad). Blots were then incubated with anti-cytochrome c (1:200, #SC-7159, Santa Cruz) and anti-heme oxygenase 1 (1:200, #SC-10789, Santa Cruz) overnight. After protein detection, both membranes were stripped (#46430, Thermo Scientific) and incubated with anti-actin (1:1,000, #A5441, Sigma-Aldrich).

In Vivo Retention Study

In order to generate luciferase-expressing MSCs, we used a third generation lentiviral vector with the general form pCCLc-MNDU3-Luciferase-PGK-eGFP-WPRE. We transduced the cells using protamine sulfate (20 μg/ml) and a quantity of virus equivalent to a multiplicity of infection of 20. Using this protocol, more than 90% of cells were eGFP positive. For cell administration, immune compromised NOD/SCID-IL2Rγ^−/− (NSG) mice were anesthetized using inhaled isoflurane and then injected with the luciferase-expressing MSCs in 20 μl of HyStem C (#G5131, BioTime) in the medial hamstring muscles. To generate the standard curve, increasing number of cells were used (from 12,500 to 100,000 cells), while 200,000 cells were injected for the retention studies. For imaging, animals were injected intraperitoneally with 100 μl of 20 mg/ml d-Luciferin Firefly (#122796, Perkin Elmer) 10 minutes before bioluminescence detection via IVIS Spectrum imaging for 5 minutes exposure time. LivingImage software was used to quantify MSCs bioluminescence (Perkin Elmer). All animal procedures were performed as approved by the Institutional Animal Care and Use Committee at UC Davis.

Glucose and Lactate Measurements

Supernatants were collected every 3 days from MSCs cultured under identical conditions to the in vitro survival assay (in 12-well plates) described above. Supernatants for glucose measurement were stored at −20°C and those for lactate at −80°C. Glucose and lactate concentrations of supernatants were determined using a Glucose Colorimetric/Fluorometric Assay Kit (#K606–100, BioVision) and a Lactate Colorimetric Assay Kit (#K607–100, BioVision), respectively, following their provided protocols.

Metabolome Analysis

One million MSCs were cultured in 225 cm² culture flasks for 48 hours in either 20% O₂ or 1% O₂ or for an additional 3 days in 1% O₂ in serum-free medium (Fig. 2A). Cells were then lifted with trypsin treatment, centrifuged at 500g, and washed once with PBS. After a second centrifugation and removal of excess liquid, tubes were snap-frozen in liquid nitrogen and stored at −80°C. Samples were then washed one more time with water (to minimize salts that could affect data acquisition), centrifuged, and submitted to the West Coast Metabolomics Center at University of California Davis. There, samples were processed and analyzed by gas chromatography—time of flight mass spectrometry (GCTOF MS) as previously described [21]). A heat map and cluster analysis were performed on the processed and normalized data using PermutMatrix software [22]. Significant differences in levels of metabolites were identified using Excel, using a paired t test.

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Figure 2.

Hypoxic preconditioning increases cell survival in vitro. (A): Schematic overview of experimental design: mesenchymal stem cells/multipotent stromal cells (MSCs) were cultured in complete culture media (supplemented with 10% FBS) for varying times in hypoxia (1% O₂). Then (time points A and B), culture media were changed to MEMa without supplements (0% FBS), with no further media changes, and incubated in 1% O₂ for up to 12 days. (B): After preincubation of MSCs for 0, 16, 48, or 96 hours in hypoxia, cells were changed to the nutrient and oxygen limited environment. Cells were counted using a hemocytometer and Trypan blue exclusion dye every 3 days. Cell survival is expressed as a percentage of remaining cells relative to day 0, after HP (n = 6). Statistical analysis was performed by ANOVA; *, p < .05. Abbreviations: FBS, fetal bovine serum; HP, hypoxic preconditioning.

HP Increases Survival of MSCs In Vitro

Given that many hypoxia-associated signals such as HIF-1α respond in a transitory manner [24] and that the therapeutic administration of MSCs is typically into tissues that are hypoxic [11], it was important to us to evaluate not only the effect of hypoxia but also the role of HP prior to new exposure to hypoxia. To mimic an environment poor in nutrients and oxygen similar to the sites where MSCs are administered in clinical applications, we performed the following set of experiments by preincubating MSCs in standard culture media in either 20% O₂ (Control) or 1% O₂ (HP). Then medium was changed to serum-free media and all cells were transferred to 1% O₂ (Fig. 2A). Since media was not changed afterward, MSCs experienced starvation due to nutrient deficiency [25]. Figure 2B shows the decline of MSC survival over time. For all conditions, no living cells could be detected after 12 days under serum deprivation. However, MSCs that received HP for 48 or 96 hours showed an approximately twofold increase in survival at 6 days under serum deprivation as compared to control MSCs (Fig. 2B), while 16 hours-HP had no effect on survival as compared to controls. These results suggest that HP for 48 hours or more transitorily promotes cell survival in environments with limited oxygen and nutrients.

To further support these results, MSCs were stained with EthD-III, which is only able to penetrate dead cells. We found that after 9 days, 48 hours HP-MSCs showed over a twofold reduction in dead cells as compared to controls (Fig. 3A, ​,3B).3B). We then measured apoptotic cells by Annexin V staining and further confirmed that after 9 days under serum deprivation, HP-MSCs were significantly less apoptotic as compared to control MSCs (Fig. 3C). To identify the possible apoptotic signaling pathway(s) involved in HP-mediated rescue of the cells under serum deprivation, we used an apoptosis proteome array. After culture for 6 or 9 days under nutrient and oxygen deficient conditions no significant differences were found in apoptosis-related signals (not shown). In contrast, on day 3, significantly lower levels of cytochrome c and heme oxygenase 1 (HO-1) were detected in HP-MSCs as compared to controls (Fig. 3D, ​,3E;3E; Supporting Information Table S1). These differences were further confirmed by Western blot (Fig. 3F). These results suggest that HP induces early changes in MSCs that promote survival under serum deprivation and hypoxia.

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Figure 3.

Hypoxic preconditioning reduces apoptosis of mesenchymal stem cells/multipotent stromal cells (MSCs) in vitro. After hypoxic preconditioning for 48 hours and culture for further 9 days, as described in Figure 3, MSCs were stained with EthD-III (dead cells) and DAPI (total nuclei). (A): Representative images of EthD-III and DAPI staining. (B): Quantification of A (n = 5). (C): Also after 9 days in culture, cells were stained with Annexin V and measured by flow cytometry (n = 5). After 3 days in culture under serum deprivation and hypoxia, total proteins were extracted and expression of apoptosis-related proteins detected using an antibody-based array. (D): Representative images. (E): Quantification of significant differences detected in the antibody array (n = 4). (F): These results were further confirmed by Western blot (n = 3). Statistical analysis was performed by paired Student’s t test; *, p < .05.

HP Enhances Retention of MSCs In Vivo

To evaluate whether HP of MSCs could enhance cell survival in vivo, we intramuscularly injected luciferase-expressing MSCs into immune deficient mice and followed their retention over time. First, to establish a correlation between IVIS-detected luminescence and cell number, we generated a standard curve by injecting increasing numbers of MSCs into the medial hamstring muscles of immune-deficient NSG mice and measured luminescence immediately thereafter (Fig. 4A). In addition, we confirmed the detection limit of this method and found that injection of 5,000 MSCs or more emit a robust signal, while the signal of 1,000 luciferase-expressing MSCs was barely detectable above background (not shown). We then addressed cellular retention with and without HP using the standard curve to determine the number of retained cells after injection over time. We found that MSCs without HP (controls) are retained for 1 week after transplantation, but rapidly decrease thereafter to undetectable levels 28 days after transplantation (Fig. 4B). In contrast, MSCs with 48 hours HP remained detectable after 28 days. Cell retention of HP-MSCs was also significantly better 14 and 21 days after transplantation as compared to control MSCs.

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Figure 4.

Hypoxic preconditioning promotes cell retention in vivo. (A): In order to establish a correlation between cell number and luminescence intensity (exerted by luciferase-expressing mesenchymal stem cells/multipotent stromal cells [MSCs]), varying amounts of cells were injected into NSG mice and measured immediately after (n = 3 animals per cell dose). (B): After preincubating MSCs for 48 hours in 20% or 1% oxygen, 200,000 cells were injected intramuscularly into NSG mice and luminescence was measured weekly (n = 14 animals per group, with MSCs derived from three different donors). For both (A) and (B), panels on the left show representative images, while average of total experiments is shown on right. Statistical analysis was performed by paired Student’s t test to each individual time point; *, p < .05.

Hypoxic Preconditioned MSCs Have More Glucose Available During Serum Deprivation In Vitro

We next sought to determine the underlying mechanism for the HP-induced survival of MSCs and hypothesized that metabolic changes play a critical role in this regard. We first measured glucose in the supernatants of MSCs and found significantly higher glucose levels in MSCs cultured in hypoxia for 96 hours (Fig. 5A), suggesting that, under this condition, MSCs consume less glucose as compared to controls. In hypoxia, most glucose is consumed through glycolysis, which leads to secretion of lactate into the supernatant. Consequently, higher lactate levels were found in MSCs cultured in hypoxia as compared to controls (Fig. 5B).

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Figure 5.

Hypoxic preconditioning reduces glucose consumption and lactate secretion. Mesenchymal stem cells/multipotent stromal cells (MSCs) were cultured for 96 hours with a single medium change after 48 hours and in 1% O₂ for the indicated time periods. Then, supernatants were collected to measure glucose (A; n = 5) and lactate (B; n = 4) as described in Materials and Methods. Then, MSCs were cultured as shown in the scheme of Figure 2A. After HP, MSCs were cultured under serum deprivation and 1% O₂ for up to 12 days with no further medium changes. Every 3 days, supernatants were collected to determine glucose (C; n = 3) and lactate (D; n = 3). Histograms to the right of (C) and (D) highlight glucose and lactate levels at day 3. Statistical analysis was performed by paired Student’s t test with Bonferroni correction; *, p < .05; **, p < .005.

After respective preconditioning, we measured glucose and lactate under serum deprivation and hypoxia (Fig. 2A). After 3 days, glucose levels were more than twofold higher in supernatants of 48 and 96 hours HP-MSCs, as compared to controls (without HP) (Fig. 5C), a result that is in line with the transitory prosurvival effect of HP. We also found increasing levels of lactate, until day 9, by which time most MSCs have died (Fig. 5D). After 3 days, lactate levels were significantly lower in 48 and 96 hours HP-MSCs as compared to controls. Taken together, these results suggest that HP reduces glucose consumption in MSCs.

This work was supported by Early Translational Grant TR2–01787 from the California Institute for Regenerative Medicine (CIRM) and NIH Transformative Grant 1R01GM099688 (J.N.). F.F., J.N., and J.B. are also funded by CIRM Disease Team Grant DR2A-05423 for Critical Limb Ischemia. We would like to thank Jane Wanyera, who, as part of the University of California Davis Teen Biotech Challenge Program and the CIRM Creativity Program, measured oxygen levels in solution from the different hypoxic incubators.