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TISSUE-SPECIFIC STEM CELLS |
Institutes of aBiochemistry and Molecular Biology,
bBiopharmaceutical Sciences, and
cClinical Medicine, National Yang-Ming University, Taipei, Taiwan;
dDepartment of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan
Key Words. Mesenchymal stem cells • Osteogenic differentiation • Mitochondria • Metabolic switch • Antioxidant enzymes
Correspondence: Correspondence: Yau-Huei Wei, Ph.D., 155 Li-Nong Street, Section 2, Peitou, Taipei 112, Taiwan. Telephone: 886-2-28267118; Fax: 886-2-28264843; e-mail: joeman{at}ym.edu.tw; or Oscar K. Lee, M.D., Ph.D., 201 Shi-Pai Road, Section 2, Taipei 112, Taiwan. Telephone: 886-2-28757557; Fax: 886-2-28757557; e-mail: kslee{at}vghtpe.gov.tw
Received on July 2, 2007;
accepted for publication on January 4, 2008.
First published online in STEM CELLS EXPRESS January 24, 2008.
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ABSTRACT |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Mitochondria are the organelles where many vital metabolic reactions take place. They are also called the power plant of mammalian cells because a majority of ATP is generated through the mitochondrial electron transport chain, in which a proton gradient is generated across the inner membrane by respiratory complexes I, III, and IV, which can drive ATP synthesis through complex V (ATP synthase) [6]. Another important feature of mitochondria is that they harbor their own genome, called mitochondrial DNA (mtDNA), which contains 16,569 base pairs of nucleotides in a circular double-stranded structure, encoding two ribosomal RNAs, 22 transfer RNAs, and 13 polypeptides [7]. However, the crosstalk between the mitochondrial and nuclear genomes in terms of the maintenance of normal cellular function, such as proliferation and differentiation, still remains elusive [8].
Apart from their bioenergetic functions, mitochondria are also the major source of endogenous reactive oxygen species (ROS) in human cells because a small portion of electrons constantly leak out from electron transport chain and contribute to the production of mitochondrial ROS, a deleterious by-product of aerobic metabolism causing oxidative damage to DNA, proteins, and lipids [9, 10]. Elevated levels of ROS can cause a variety of human diseases, such as cardiovascular diseases, ischemia/reperfusion injuries, neurological disorders, diabetes, and cancer, as a consequence of tissue damages. To overcome the detrimental effects of ROS, there is an array of defense systems, including enzymatic and nonenzymatic antioxidants in intra- and extracellular spaces to protect cells from the attack of ROS [11]. The leaked-out electrons of electron transport chain react with oxygen to form superoxide anions (O2
), which can be converted by superoxide dismutase (SOD) to hydrogen peroxide (H2O2), followed by further decomposition to H2O and O2 by catalase and glutathione peroxidase [12, 13]. Since ROS also serves as a second messenger in signal transduction, production of ROS and the activities of antioxidant enzymes must be tightly controlled to maintain the homeostasis of the intracellular redox status so that proper ROS-mediated signaling can take place without elevating intracellular oxidative stress.
The density and activity of mitochondria vary in different types of terminally differentiated cells. However, little is known regarding how this diversity is controlled during the developmental processes of an organism from a single embryo. Lack of clear understanding of the intergenomic communication in this process necessitates the investigation of the mechanisms underlying the regulation of mitochondrial biogenesis during cellular differentiation and maturation. Therefore, we took advantage of the well-established osteogenic differentiation system of hMSCs to dissect changes in cellular bioenergetic functions [14]. We hypothesized that mitochondrial functions are altered in response to osteogenic differentiation signals. In this study, we found that during osteogenic differentiation of hMSCs, mtDNA copy number, protein subunits of respiratory enzymes, oxygen consumption rate, intracellular ATP level, and antioxidant enzymes are positively regulated, whereas the intracellular ROS is suppressed, demonstrating a well-coordinated process involving upregulation of the biogenesis and respiratory function of mitochondria and expression of antioxidant enzymes.
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MATERIALS AND METHODS |
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Alkaline Phosphatase Assay
For alkaline phosphatase (ALP) staining, cells in 35-mm dishes were fixed with 4% paraformaldehyde and stained with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Sigma-Aldrich). ALP activity was assayed as previously described [17]. Cells in 96-well plates were lysed with 0.05% SDS at 37°C for 10 minutes and incubated in a solution containing 8 mM 4-nitrophenyl phosphate and 2 mM MgCl2 in 2-amino-2-methyl-1-propanol (Sigma-Aldrich) for 30 minutes in the dark at 37°C. The reaction was stopped with 0.02 N NaOH, and the absorbance at 405 nm was measured by an enzyme-linked immunosorbent assay (ELISA) reader (Power Wave HT 340; Bio-Tek Instruments, Inc., Winooski, VT, http://www.biotek.com). To examine the effect of H2O2 or oligomycin on osteogenic differentiation, hMSCs were treated 2 days post-osteogenic induction, and the ALP activity was determined on the 5th day of induction.
RNA Extraction and Reverse Transcription-Quantitative Polymerase Chain Reaction
RNA was extracted with TRI Reagent (Sigma-Aldrich), and 5 µg of RNA was reverse-transcribed to cDNA with the Ready-to-Go reverse transcription-quantitative-polymerase chain reaction (RT-QPCR) kit (Amersham Biosciences, Uppsala, Sweden, http://www.amersham.com). Quantitative PCR analysis was performed using the LightCycler TaqMan Master kit (Roche Applied Sciences, Mannheim, Germany, http://www.roche-applied-science.com) according to the manufacturer's instructions. Gene expression levels were normalized by the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Information about the primer pairs and probes is listed in supplemental online Table 1.
Measurement of Mitochondrial Mass
Mitochondrial mass was measured as previously described [18]. Cells were incubated in fresh medium with 2.5 µM nonyl acridine orange (NAO; Molecular Probes, Eugene, OR, http://probes.invitrogen.com) for 10 minutes at 25°C in the dark and harvested in a solution containing 5 mM KCl, 140 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 5 mM HEPES buffer (pH 7.4). The fluorescence intensity of 10,000 cells was recorded on a flow cytometer (model EPICS XL-MCL; Beckman Coulter, Miami, FL, http://www.beckmancoulter.com) with the excitation wavelength at 488 nm and emission wavelength at 535 nm.
Determination of Relative mtDNA Copy Number
An aliquot of 50 ng of DNA was subjected to quantitative PCR using LightCycler-FastStar DNA Master SYBR Green I kit (Roche Applied Sciences). DNA fragments of NADH dehydrogenase subunit 1 (ND1) gene (mtDNA-encoded) and β-actin gene (nuclear DNA-encoded, served as internal control) were amplified with specific primer pairs (supplemental online Table 1), respectively. The relative mtDNA copy number was measured by normalization of the crossing points in quantitative PCR curves between ND1 and β-actin genes using the RelQuant software (Roche Applied Sciences). PCR was performed as follows: 1 cycle of hot start at 95°C for 10 minutes and 35 cycles of 5-second denaturation at 95°C, 10-second annealing at 58°C, and 20-second extension at 72°C.
Western Blot Analysis
An aliquot of 25 µg of proteins was separated on a 12% SDS-polyacrylamide gel electrophoresis gel and blotted onto a polyvinylidene difluoride membrane (Amersham Biosciences). Nonspecific bindings were blocked by 3% skim milk in Tris-buffered saline tween 20 buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.4), and the membrane was blotted with indicated primary antibodies (supplemental online Table 2). After incubation with a horseradish peroxidase-conjugated secondary antibody, the protein intensity was determined by an enhanced chemiluminescence reagent (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com).
Measurement of Oxygen Consumption
Oxygen consumption rate was measured by the 782 Oxygen Meter (Strathkelvin Instruments, Motherwell, U.K., http://www.strathkelvin.com). An aliquot of 5 x 105 cells was incubated on ice for 10 minutes in 330 µl of assay buffer (125 mM sucrose, 65 mM KCl, 2 mM MgCl2, 20 mM phosphate buffer, pH 7.2) containing 0.0003% digitonin (Sigma-Aldrich) to permeabilize the mitochondrial outer membrane. The cell suspension was then transferred into the incubation chamber, and the oxygen consumption rate was measured after injection of 6 mM succinate into the chamber as the electron donor.
Measurement of Lactate Production Rate
Lactate production rate was measured by a Lactate Reagent kit (Trinity Biotech plc., Bray, Ireland, http://www.trinitybiotech.com). Cells in six-well plates were replenished with fresh medium and incubated for 8 hours. An aliquot of 10 µl of medium was then transferred to a 96-well plate to mix with the Lactate Reagent, and the absorbance at 540 nm was measured by an ELISA reader (Power Wave HT 340). The absorbance was then normalized by total cell number and divided by the time of incubation.
Measurement of Intracellular ATP Content
Intracellular ATP level was measured by the Bioluminescent Somatic Cell Assay Kit (Sigma-Aldrich). An aliquot of 50 µl of viable cell suspension was mixed with 150 µl of Somatic Cell Releasing Reagent to release the intracellular ATP. Half of the mixture was then transferred into a black OptiPlate-96F 96-well plate (Packard Biosciences, Perkin-Elmer) containing 100 µl of ATP Assay Mix, and the luminescence intensity was measured by the Victor2 1420 Multilabel Counter (PerkinElmer Life and Analytical Sciences). The luminescence intensity was then divided by total cell number.
alamarBlue Cell Viability Assay
Cells in 96-well plates were incubated with fresh medium containing 1 x alamarBlue cell viability assay reagent (AbD Serotec, Oxford, U.K., http://www.ab-direct.com) at 37°C for 4 hours. The fluorescence intensity was measured by the Victor2 1420 Multilabel Counter (PerkinElmer Life and Analytical Sciences) with the excitation wavelength at 538 nm and emission wavelength at 590 nm.
Determination of Intracellular ROS
For measurement of H2O2 and O2 levels, cells were incubated in a medium containing 40 µM 2',7'-dichlorodihydrofluorescein diacetate and 5 µg/ml hydroethidine (Molecular Probes), respectively, at 37°C in the dark for 10 minutes. Cells were then resuspended in 50 mM HEPES buffer, and the fluorescence intensity of 10,000 cells was recorded on a flow cytometer (model EPICS XL-MCL) with the excitation wavelength at 488 nm and emission wavelengths at 535 and 580 nm for measurement of H2O2 and O2, respectively.
Activity Assay of Catalase and Total SOD
Catalase activity was determined by monitoring the rate of decomposition of H2O2 from the decrease in absorbance at 240 nm [10]. Total SOD activity was assayed by monitoring nitroblue tetrazolium (NBT) reduction according to Spitz and Oberley [19] with some modifications. SOD inhibits NBT reduction caused by O2 in the aerobic xanthine/xanthine oxidase system, and changes of absorbance at 560 nm within 2 minutes were recorded. One unit of SOD is defined as the amount of enzyme that causes 45% inhibition of NBT reduction under the assay conditions described [10].
Statistical Analysis
Statistical analysis was performed using the SPSS program for Windows, Standard Version (version 10.0.1, SPSS Inc., Chicago, http://www.SPSS.com). The data are presented as mean ± SD of the results from three independent experiments. The significance level was determined by nonparametric Mann-Whitney U test. A difference was considered to be statistically significant at p < .05.
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RESULTS |
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Upregulation of Mitochondrial Biogenesis-Associated Genes
To further explore the underlying mechanism involved in the enhancement of mitochondrial respiratory function, we examined the mRNA expression levels of three crucial genes associated with mitochondrial biogenesis including mitochondrial transcription factor A (mtTFA), DNA polymerase 
(Pol ), and peroxisome proliferator-activated receptor coactivator-1
(PGC-1). By real-time PCR, we observed a gradual increase of mtTFA and Pol expression along with the osteogenic differentiation, and the changes reached a plateau at around day 21 (Fig. 2D; mtTFA, 4.6 ± 1.1; Pol , 4.9 ± 0.9-fold). PGC-1 showed a more dynamic expression pattern, with a peak expression level on day 7 (14.8 ± 5.9-fold).
Decrease in the Expression of Glycolytic Enzymes and Lactate Production Rate During Osteogenic Induction
Since the mitochondrial respiratory activity was augmented, it raised another question as to whether the glycolytic activity was changed. By Western blot analysis, we found a significant decrease in the protein levels of glucophosphate isomerase (GPI; 0.59 ± 0.13-fold) and phosphofructokinase (PFK; 0.63 ± 0.20-fold) in ost2w cells (Fig. 3A). On the other hand, the protein level of pyruvate dehydrogenase (PDH), an enzyme that is responsible for converting pyruvate into acetyl CoA to enter the tricarboxylic acid cycle and aerobic metabolism, was dramatically increased (2.19 ± 1.31-fold). In addition, pyruvate dehydrogenase kinase (PDK), which can phosphorylate PDH to inhibit its activity, was found to be significantly decreased (<0.10-fold). We also measured the lactate production rate and found that undifferentiated hMSCs had a higher lactate production rate compared with ost2w (Fig. 3B; 941.6 ± 79.0 vs. 503.2 ± 32.3 ng per 104 cells per hour; p < .01), indicating profound anaerobic glycolytic metabolism in hMSCs. Consequently, the intracellular ATP content of hMSCs was measured, and we found that intracellular ATP content was initially decreased (17.8 ± 2.9 pmol per cell) on the 2nd day of induction in comparison with undifferentiated hMSCs (23.6 ± 2.9 pmol per cell), was subsequently increased to 1.5 times of that of control (35.7 ± 4.6 pmol per cell) on the 4th day of induction, and was maintained stably thereafter (Fig. 3C).
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were dramatically reduced as early as the 2nd day of osteogenic induction (Fig. 5A; H2O2, 32.5% ± 3.9%; O2, 72.0% ± 7.6% of undifferentiated hMSCs; p < .05). However, as osteogenic differentiation went on, gradual rebound of H2O2 and O2 was noted. H2O2 was recovered to 62.8% ± 18.2%, and the O2 level was slightly higher than that of undifferentiated hMSCs (113.7% ± 13.0%) after 28 days of induction. The unexpected decline of intracellular ROS prompted us to ask whether this phenomenon was caused by the changes in the antioxidant capacity of hMSCs during differentiation. We found that protein levels of MnSOD and catalase were significantly upregulated in a time-dependent manner during osteogenic differentiation, but no such change was observed in copper/zinc-dependent superoxide dismutase (Fig. 5B). Quantification by densitometry revealed that the amounts of catalase and MnSOD were increased to approximately threefold and fourfold, respectively, after 14 days of induction (Fig. 5C). We also measured the levels of other proteins of the antioxidant defense system, including glutathione reductase, glutathione peroxidase (GPx), peroxiredoxin-I (Prx-I), peroxiredoxin-III (Prx-III), thioredoxin-I (Trx-I), and thioredoxin reductase (TrxR) by Western blot analysis, but no significant differences in any of these proteins were noted (data not shown). These results indicate that during osteogenic differentiation, only a few enzymes of the antioxidant defense system are strongly upregulated in hMSCs to reduce endogenous ROS.
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, significant reduction in the viability of hMSCs was observed in both treatments (Fig. 6C, 6D). Upon treatment with concentrations as high as 750 µM H2O2, the viability of hMSCs was 50.8% ± 1.5%, whereas that of ost2w was 97.1% ± 16.4%. Upon treatment with 25 µM menadione, the viability of hMSCs was 15.2% ± 4.3%, whereas that of ost2w was 37.7% ± 4.0%. These results indicate that the antioxidative capacity was increased during the osteogenic differentiation of hMSCs.
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DISCUSSION |
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The intergenomic communication between mitochondrial and nuclear genomes in stem cells is complicated and remains elusive [21]. The increase in the copy number of mtDNA in response to osteogenic induction without concomitant increase of mitochondrial mass (Fig. 2A) indicates that the regulations of mtDNA copy number and mitochondrial mass are not coupled during differentiation. Nevertheless, in our study, mitochondrial mass was measured by a commonly used fluorescent dye, NAO, which binds specifically to cardiolipin on the mitochondrial inner membrane. It might be possible that during osteogenic differentiation of hMSCs, mitochondrial biogenesis was intensified in conjunction with minor changes in the cardiolipin content, but the protein levels of respiratory enzyme complexes, oxygen consumption rate, and mRNA levels of three mitochondrial biogenesis-associated genes, Pol , mtTFA, and PGC-1 [22–24], were increased (Fig. 2). One possible reason is that during osteogenic differentiation of hMSCs, mitochondrial ultrastructure is altered to form more cristae protruding into the matrix to accommodate more respiratory enzyme complexes. Lonergan et al. [5] showed that the stem cell property is associated with the perinuclear arrangement of mitochondria in adult monkey stromal stem cells. Consistently, we observed perinuclear distribution of mitochondria in the early phase of osteogenic induction, and an evenly distributed pattern was resumed after 2 weeks of differentiation (unpublished data).
Although oxidative phosphorylation is the most efficient way to generate ATP, it is not always the best way for cells to fuel themselves. Early in the 1950s, Warburg [25] formally proposed that cancer cells rely primarily on anaerobic glycolysis instead of oxidative phosphorylation. This phenomenon is termed Warburg effect. This has been extensively studied to understand the metabolic aberrations of cancer cells. Similarly, the metabolic pattern of stem cells may also be different from that of terminally differentiated cells. Kondoh et al. [4] demonstrated that the highly proliferative capacity of murine ESCs is closely associated with high activity of glycolytic enzymes, elevated glycolytic flux, and low mitochondrial oxygen consumption. In the present study, we observed that hMSCs expressed higher levels of glycolytic enzymes, such as GPI, PFK, and PDK, and a lower level of PDH (Fig. 3A). Another index for anaerobic metabolism, lactate production rate, was also higher in undifferentiated hMSCs than in ost2w (Fig. 3B), indicating a high glycolytic activity. Together with the findings of the increase of mitochondrial biogenesis, it is evident that an energy production switch from anaerobic glycolysis to mitochondrial respiration and oxidative phosphorylation occurred during the osteogenic differentiation of hMSCs. An overall increase of intracellular ATP content (Fig. 3C) indicates that energy burst is essential for the hMSCs to go through the differentiation process. In addition, hMSCs were more vulnerable to glycolytic inhibitor-induced cell death (Fig. 4A, 4B), which suggests that hMSCs indeed rely more on glycolysis in comparison with the differentiated osteoblasts, ost2w. The lower levels of ATP content (Fig. 3C) and mitochondrial biogenesis of hMSCs may reflect the quiescence-maintaining property of stem cells. It seems that hMSCs prefer to produce energy by glycolysis to avoid the production of ROS, the deleterious byproducts of aerobic metabolism.
Mitochondria are the major source of endogenous ROS in human cells. The electron leaked out from the electron transport chain contributes to the production of mitochondrial ROS. In the present study, intracellular H2O2 and O2 levels were significantly reduced (Fig. 5A), although mitochondrial respiration was enhanced during osteogenic differentiation of hMSCs. The possibility that the reduction of intracellular ROS was due to ascorbic acid added in the osteogenic induction medium was excluded by the observation that ROS was not decreased simply by adding ascorbic acid to the culture medium (day 6, 89.5% ± 5.6%). On the contrary, the intracellular H2O2 levels in cells treated with dexamethasone and β-glycerol phosphate without ascorbic acid was lower (day 6, 61.4% ± 9.8%) than that of cells treated with ascorbic acid alone. However, the H2O2 level was not as low as that found when osteogenic induction was induced (Fig. 5A; day 6, 40.3% ± 3.4%). These findings suggest that the antioxidative effects are mainly contributed by differentiation rather than by ascorbic acid. On the other hand, the time-dependent upregulation of catalase and MnSOD upon osteogenic induction (Fig. 5B, 5C) was closely correlated with the decrease of intracellular ROS. Results from measurement of catalase and total SOD activities (Fig. 6A, 6B), as well as cell viability assay after exogenous ROS treatment (Fig. 6C, 6D) demonstrate that differentiated osteoblasts had relatively higher antioxidative capacity. This finding suggests that during osteogenic differentiation, a coordinated regulation between mitochondrial biogenesis and antioxidant defense systems is orchestrated to prevent the accumulation of ROS when aerobic metabolism of mitochondria becomes dominant. Another effective regulator, uncoupling protein 2 (UCP2), might also contribute to the alterations of mitochondrial ATP synthesis and ROS generation [26]. We found that the mRNA expression level of UCP2 was increased, with a peak level on day 21 after osteogenic induction (data not shown). Additional experiments are needed to determine whether altered expression of UCP2 is related to the dramatic changes of mitochondrial respiratory function and intracellular ROS in the differentiation process of hMSCs.
Recent studies have indicated that cell differentiation is also influenced by ROS. Su et al. [27] showed that ROS enhanced vascular smooth muscle cell differentiation through the p38/MAPK-dependent pathway. On the contrary, Mody et al. [28] reported that ROS inhibited the differentiation of bone preosteoblast cell line MC3T3-E1. We found that sublethal doses of exogenous H2O2 significantly reduced the ALP activity and the mRNA expression level of the osteoblast-specific maker gene Cbfa-1 (Fig. 7A, 7C). On the other hand, oligomycin also reduced the ALP activity and Cbfa-1 expression (Fig. 7B, 7C), suggesting the importance of mitochondrial respiratory function during osteogenic differentiation. However, since oligomycin not only inhibits ATP synthase activity but also increases the production of intracellular ROS, additional studies need to be carried out to delineate whether oligomycin or ROS would be the major inhibitor to osteogenic differentiation of hMSCs. We speculate that a well-organized regulatory circuit must be turned on to control mitochondrial biogenesis and antioxidative defense systems to create a proper intracellular environment for hMSC differentiation.
We observed significant decrease in lactate production rate and increase in the protein expression of MnSOD 7 days after osteogenic induction of hMSCs from five different donors (supplemental online Fig. 1). These observations support the possibility that there is coordination between increase of mitochondrial respiration and upregulation of antioxidant enzymes during osteogenic differentiation of hMSCs.
It is also important to identify the messengers that orchestrate the coordinated expression of the genes encoding respiratory enzymes and antioxidant enzymes. Recently, St-Pierre et al. [29] demonstrated that PGC-1 is required for not only mitochondrial biogenesis but also the induction of several ROS-detoxifying enzymes, including MnSOD and GPx-1, after exposure of the cells to an oxidative stressor. We also observed an upregulation of PGC-1 during osteogenic differentiation of hMSCs. Further effort should be made to investigate whether PGC-1 is regulated by osteogenesis-specific genes to coordinate mitochondrial biogenesis and antioxidant enzyme expression. Interestingly, we noticed that the mRNA expression levels of mtTFA, MnSOD, and catalase were also upregulated during hepatogenic differentiation, whereas only mtTFA and catalase levels (and not MnSOD levels) were increased during chondrogenic differentiation of hMSCs (unpublished data). These results suggest that the coordinated changes in mitochondrial biogenesis and antioxidant enzymes are a general regulatory mechanism during differentiation of hMSCs, but lineage-specific gene regulation still exists.
In summary, we demonstrated that alterations in mitochondrial biogenesis and antioxidant enzymes are well coordinated during osteogenic differentiation of hMSCs. Understanding the roles of mitochondria and ROS during cell differentiation will facilitate the optimization of in vitro differentiation protocols by adjusting biochemical properties such as energy production or redox status of stem cells for better design of cell therapy. Besides, the energy production profile or bioenergetic signature dictated by mitochondrial biogenesis may also serve as an index to identify stem cells of better quality in a given cell pool.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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