Cell Technologies in Biology and Medicine, No. 1, May, 2014
167
Effect of Hypoxia on Porphyrin Metabolism in Bone Marrow Mesenchymal Stem Cells A. G. Poleshko, E. S. Lobanok, and I. D. Volotovskii
Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 57-62, January, 2014 Original article submitted March 25, 2013 Under hypoxic conditions, aminolevulinic acid-induced accumulation of porphyrin pigments and increase in heme content was observed in bone marrow mesenchymal stem cells. The expression of transferrin receptor CD71 responsible for Fe2+ transport into the cell was also enhanced. Blockade of porphyrin-transporting protein ABCG2 with fumitremorgin C under conditions of normoxia and hypoxia induced accumulation of porphyrin pigments; in hypoxia, these changes were more pronounced. Key Words: bone marrow mesenchymal stem cells; hypoxia; heme; porphyrin pigments, transporter protein ABCG2 Regenerative therapy with mesenchymal stem cells (MSC) is a promising trend in the treatment of various human diseases [1]. Protocols of isolation, culturing, and expansion of MSC for transplantation to the injuries organs and tissues have been developed. Under optimal conditions, MSC can pass tens proliferation cycles without losing their multipotency [1,9]. An important problem of cell biology is improvement of the technique of express-expansion of the cell material characterized by high viability and proliferative activity. The functional state of MSC in culture is controlled by external factors including oxygen concentration in the culture medium [4,18]. Adaptation to low oxygen content in the medium is accompanied by activation of specifi c hypoxiainduced mechanisms of genetic control that indirectly (through activation-suppression of the target genes) optimized metabolic processes that ensure homeostasis and maintain cell viability [13,28]. It was hypothesized that oxygen concentrations ranging from 1 to 8%, rather than atmospheric concentration of 21% commonly used for MSC culturing, are favorable for the processes in the cell culture; these Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus, Minsk, Belarus. Address for correspondence: [email protected]. A. G. Poleshko
concentrations are observed in tissues and organs of living organisms [3,6], including the bone marrow (BM) and adipose tissue [18]. Porphyrin metabolism is an important metabolic process that reacts to variations in oxygen content in the medium [10,12,20]. Oxygen deficiency leads to accumulation of heme metabolites in the cell, which can lead to pathological changes and damage to the cells [14-16]. In previous studies, Verapamil-sensitive heme- and porphyrin-transporting proteins were described: HCP1 (heme carrier protein 1), FLVCR (feline leukemia virus subgroup C receptor), ABCG6 (ATP-binding cassette subfamily G member 6), ABCG2 (ATP-binding cassette sub-family G member 2) [11,22]. These proteins are thought to be important for the maintenance of the concentration and balance of heme/porphyrins at a constant and optimal level. A special role in this process is played by ATP-binding protein ABCG2 [24,26]. ABCG2 protein is expressed in most animal cells; it is more abundant in the liver, intestinal epithelium, placenta, components of the blood-brain barrier (blood capillary endothelial cells and astrocytes), stem cells of various types, and tumor cells with characterized by pronounced xenobiotic-excretion capacity, which determines their chemical resistance [23]. ABCG2 is also a protein marker of side population cells characterized
0007-4888/14/15710167 © 2014 Springer Science+Business Media New York
168 by the ability of excreting fluorescent dyes that specifically bind to DNA into extracellular medium [29]. The functions of ABCG2 protein in MSC remain unknown. It was hypothesized that ABCG2 can perform a protective role and improve survival of stem cells under hypoxic conditions [14,19] via transport of porphyrin pigments from the cell into the extracellular medium [26]. Here we studied the influence of hypoxia on porphyrin metabolism in MSC from rat BM and the mechanisms of its maintenance under conditions of oxygen deficiency.
MATERIALS AND METHODS The study was performed on BM MSC from 3-5-month-old outbred albino rats. Cell culture. For MSC isolation, BM bar was extruded from the femur and tibia with α-MEM containing 10% embryonic calf serum (ECS, HyClone), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The material was disaggregated to single-cell suspension that was resuspended and seeded to culture flasks (Sarstedt) in a concentration of 1-2×106 cells/cm2. The medium with non-adherent cells was removed in 24 h, adherent cells were washed with 0.155 M PBS (pH 7.4), and fresh culture medium was added. The medium was replaced every 3 days. After attaining 70-80% confluence, the cells were treated with 0.25-0.02% trypsinEDTA (Stem Cell Technology) and transferred to new flasks. The cells were counted in a Goryaev chamber. Passage 2-3 cells were used in the study. Phenotyping showed that 94-96% cells in the studied cultures had CD44+/CD90+ phenotype typical of BM MSC. The content of CD45+ cells did not exceed 2%. Hypoxia modeling. In 1 day after subculturing, the medium with 10% ECS was replaced with the growth medium of the same composition but 2% ECS. Then, some cultures were transferred to hypoxic CO2 incubator for culturing at 5% oxygen (5% CO2, 5% O2, 90% N2) and other cultures were left under standard conditions (5% СО2, 95% atmospheric air). The cells were cultured under hypoxic conditions for 72 h, because shorter expansion time was insufficient for obtaining required cell biomass (70-80% confluence was attained only after 3 days). Aminolevulinic acid concentration [19]. For measurement of the intracellular content of aminolevulinic acid (ALA), 0.5 ml 20% trichloroacetic acid was added to 1 ml cell suspension (107 cells/ml); the samples were boiled in a water bath for 15 min, cooled at room temperature, and centrifuged for 10 min at 1500 rpm; the supernatant was transferred to a new tube, while the precipitate was resuspended in 1 ml 0.1 M acetate buffer (pH 4.6), resuspended, and re-centrifuged for 10
Cell Technologies in Biology and Medicine, No. 1, May, 2014
min at 1500 rpm. The supernatants were pooled and 1 drop of acetylacetone was added. The samples were incubated for 15 min in a water bath, Ehrlich reagent was added (1:1), and optical density was measured in 10 min on a Spekol 11 photometer at λ=155 nm. ALA concentration was measured by the calibration curve. Content of porphyrin pigments [5]. It was previously demonstrated, that maximum induction of prophynogenesis in thymocytes, peripheral blood lymphocytes, splenocytes, and Molt lymphoblasts was observed after addition of 0.8 mM ALA [2]. Similar effect was observed in BM MSC. The cells harvested from plastic (105 cells/ml) were transferred to PBS and ALA (Sigma-Aldrich) was added to a final concentration of 0.8 mM. The samples were incubated at 37oC for 4 h and the content of ALA-induced porphyrin pigments in the cell suspension was measured by their fluorescence on a Solar СМ2203 spectrofluorometer at λex=405 nm and λem=636 nm. Porphyrin content was expressed in relative units per 106 cells. Heme content [27]. Heme concentration in MSC was measured by the content of protoporphyrin IX formed in cells after 40-min boiling in 2 M oxalic acid. Calibration curve was constructed using hemin solutions containing 1% BSA. Protoporphyrin IX fluorescence was measured on a Solar CM2203 spectrofluorometer at λex=405 nm and λem=602 nm. Expression of transferring receptor CD71 [27]. For evaluation of the expression of CD71 protein, MSC (105 cells) resuspended in 20 μl PBS with 1% ECS were incubated with FITC-labeled antibodies to CD71 (1:20; Termo Scientific) for 30 min at room temperature, washed twice with non-diluted commercial ECS and the pellet was resuspended in 200 μl PBS. The cells were analyzed on a FACSCanto II flow cytofluorometer (Becton Dickinson) in the FITC channel; FITC-CD71 fluorescence intensity histograms were analyzed using DIVA 6.0 software. Samples containing unspecific FITC-labeled immunoglobulin of the same isotype as the antibody to the test marker diluted 1:20 were used as the control. MSC treatment with fumitremorgin C. Fumitremorgin C (FTC) is a functional blocker of porphyrintransporter protein ABCG2 [7]. FTC (Sigma-Aldrich) in a concentration of 10 μM [26] was added to the suspension of MSC in PBS and incubated for 1.5 h at 37oC in the dark. After incubation, the cell suspension was centrifuged for 5 min at 1500 rpm, the supernatant was separated from the pellet (cells), and porphyrin content was evaluated by their fluorescence, which reflected the efficiency of transmembrane transport of pigments. Statistical analysis. The data were processed statistically using Statistica 6.0 software. The data are presented as the mean±standard deviation. The groups
A. G. Poleshko, E. S. Lobanok, and I. D. Volotovskii
were compared using Mann–Whitney U test. The differences were significant at p
167
Effect of Hypoxia on Porphyrin Metabolism in Bone Marrow Mesenchymal Stem Cells A. G. Poleshko, E. S. Lobanok, and I. D. Volotovskii
Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 57-62, January, 2014 Original article submitted March 25, 2013 Under hypoxic conditions, aminolevulinic acid-induced accumulation of porphyrin pigments and increase in heme content was observed in bone marrow mesenchymal stem cells. The expression of transferrin receptor CD71 responsible for Fe2+ transport into the cell was also enhanced. Blockade of porphyrin-transporting protein ABCG2 with fumitremorgin C under conditions of normoxia and hypoxia induced accumulation of porphyrin pigments; in hypoxia, these changes were more pronounced. Key Words: bone marrow mesenchymal stem cells; hypoxia; heme; porphyrin pigments, transporter protein ABCG2 Regenerative therapy with mesenchymal stem cells (MSC) is a promising trend in the treatment of various human diseases [1]. Protocols of isolation, culturing, and expansion of MSC for transplantation to the injuries organs and tissues have been developed. Under optimal conditions, MSC can pass tens proliferation cycles without losing their multipotency [1,9]. An important problem of cell biology is improvement of the technique of express-expansion of the cell material characterized by high viability and proliferative activity. The functional state of MSC in culture is controlled by external factors including oxygen concentration in the culture medium [4,18]. Adaptation to low oxygen content in the medium is accompanied by activation of specifi c hypoxiainduced mechanisms of genetic control that indirectly (through activation-suppression of the target genes) optimized metabolic processes that ensure homeostasis and maintain cell viability [13,28]. It was hypothesized that oxygen concentrations ranging from 1 to 8%, rather than atmospheric concentration of 21% commonly used for MSC culturing, are favorable for the processes in the cell culture; these Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus, Minsk, Belarus. Address for correspondence: [email protected]. A. G. Poleshko
concentrations are observed in tissues and organs of living organisms [3,6], including the bone marrow (BM) and adipose tissue [18]. Porphyrin metabolism is an important metabolic process that reacts to variations in oxygen content in the medium [10,12,20]. Oxygen deficiency leads to accumulation of heme metabolites in the cell, which can lead to pathological changes and damage to the cells [14-16]. In previous studies, Verapamil-sensitive heme- and porphyrin-transporting proteins were described: HCP1 (heme carrier protein 1), FLVCR (feline leukemia virus subgroup C receptor), ABCG6 (ATP-binding cassette subfamily G member 6), ABCG2 (ATP-binding cassette sub-family G member 2) [11,22]. These proteins are thought to be important for the maintenance of the concentration and balance of heme/porphyrins at a constant and optimal level. A special role in this process is played by ATP-binding protein ABCG2 [24,26]. ABCG2 protein is expressed in most animal cells; it is more abundant in the liver, intestinal epithelium, placenta, components of the blood-brain barrier (blood capillary endothelial cells and astrocytes), stem cells of various types, and tumor cells with characterized by pronounced xenobiotic-excretion capacity, which determines their chemical resistance [23]. ABCG2 is also a protein marker of side population cells characterized
0007-4888/14/15710167 © 2014 Springer Science+Business Media New York
168 by the ability of excreting fluorescent dyes that specifically bind to DNA into extracellular medium [29]. The functions of ABCG2 protein in MSC remain unknown. It was hypothesized that ABCG2 can perform a protective role and improve survival of stem cells under hypoxic conditions [14,19] via transport of porphyrin pigments from the cell into the extracellular medium [26]. Here we studied the influence of hypoxia on porphyrin metabolism in MSC from rat BM and the mechanisms of its maintenance under conditions of oxygen deficiency.
MATERIALS AND METHODS The study was performed on BM MSC from 3-5-month-old outbred albino rats. Cell culture. For MSC isolation, BM bar was extruded from the femur and tibia with α-MEM containing 10% embryonic calf serum (ECS, HyClone), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The material was disaggregated to single-cell suspension that was resuspended and seeded to culture flasks (Sarstedt) in a concentration of 1-2×106 cells/cm2. The medium with non-adherent cells was removed in 24 h, adherent cells were washed with 0.155 M PBS (pH 7.4), and fresh culture medium was added. The medium was replaced every 3 days. After attaining 70-80% confluence, the cells were treated with 0.25-0.02% trypsinEDTA (Stem Cell Technology) and transferred to new flasks. The cells were counted in a Goryaev chamber. Passage 2-3 cells were used in the study. Phenotyping showed that 94-96% cells in the studied cultures had CD44+/CD90+ phenotype typical of BM MSC. The content of CD45+ cells did not exceed 2%. Hypoxia modeling. In 1 day after subculturing, the medium with 10% ECS was replaced with the growth medium of the same composition but 2% ECS. Then, some cultures were transferred to hypoxic CO2 incubator for culturing at 5% oxygen (5% CO2, 5% O2, 90% N2) and other cultures were left under standard conditions (5% СО2, 95% atmospheric air). The cells were cultured under hypoxic conditions for 72 h, because shorter expansion time was insufficient for obtaining required cell biomass (70-80% confluence was attained only after 3 days). Aminolevulinic acid concentration [19]. For measurement of the intracellular content of aminolevulinic acid (ALA), 0.5 ml 20% trichloroacetic acid was added to 1 ml cell suspension (107 cells/ml); the samples were boiled in a water bath for 15 min, cooled at room temperature, and centrifuged for 10 min at 1500 rpm; the supernatant was transferred to a new tube, while the precipitate was resuspended in 1 ml 0.1 M acetate buffer (pH 4.6), resuspended, and re-centrifuged for 10
Cell Technologies in Biology and Medicine, No. 1, May, 2014
min at 1500 rpm. The supernatants were pooled and 1 drop of acetylacetone was added. The samples were incubated for 15 min in a water bath, Ehrlich reagent was added (1:1), and optical density was measured in 10 min on a Spekol 11 photometer at λ=155 nm. ALA concentration was measured by the calibration curve. Content of porphyrin pigments [5]. It was previously demonstrated, that maximum induction of prophynogenesis in thymocytes, peripheral blood lymphocytes, splenocytes, and Molt lymphoblasts was observed after addition of 0.8 mM ALA [2]. Similar effect was observed in BM MSC. The cells harvested from plastic (105 cells/ml) were transferred to PBS and ALA (Sigma-Aldrich) was added to a final concentration of 0.8 mM. The samples were incubated at 37oC for 4 h and the content of ALA-induced porphyrin pigments in the cell suspension was measured by their fluorescence on a Solar СМ2203 spectrofluorometer at λex=405 nm and λem=636 nm. Porphyrin content was expressed in relative units per 106 cells. Heme content [27]. Heme concentration in MSC was measured by the content of protoporphyrin IX formed in cells after 40-min boiling in 2 M oxalic acid. Calibration curve was constructed using hemin solutions containing 1% BSA. Protoporphyrin IX fluorescence was measured on a Solar CM2203 spectrofluorometer at λex=405 nm and λem=602 nm. Expression of transferring receptor CD71 [27]. For evaluation of the expression of CD71 protein, MSC (105 cells) resuspended in 20 μl PBS with 1% ECS were incubated with FITC-labeled antibodies to CD71 (1:20; Termo Scientific) for 30 min at room temperature, washed twice with non-diluted commercial ECS and the pellet was resuspended in 200 μl PBS. The cells were analyzed on a FACSCanto II flow cytofluorometer (Becton Dickinson) in the FITC channel; FITC-CD71 fluorescence intensity histograms were analyzed using DIVA 6.0 software. Samples containing unspecific FITC-labeled immunoglobulin of the same isotype as the antibody to the test marker diluted 1:20 were used as the control. MSC treatment with fumitremorgin C. Fumitremorgin C (FTC) is a functional blocker of porphyrintransporter protein ABCG2 [7]. FTC (Sigma-Aldrich) in a concentration of 10 μM [26] was added to the suspension of MSC in PBS and incubated for 1.5 h at 37oC in the dark. After incubation, the cell suspension was centrifuged for 5 min at 1500 rpm, the supernatant was separated from the pellet (cells), and porphyrin content was evaluated by their fluorescence, which reflected the efficiency of transmembrane transport of pigments. Statistical analysis. The data were processed statistically using Statistica 6.0 software. The data are presented as the mean±standard deviation. The groups
A. G. Poleshko, E. S. Lobanok, and I. D. Volotovskii
were compared using Mann–Whitney U test. The differences were significant at p
