Physiological and hypoxic oxygen concentration differentially regulates human c-Kit⁺ cardiac stem cell proliferation and migration

Physiological (5%) oxygen is optimal for proliferation and migration of c-kit⁺ cardiac stem cells (CSCs) compared with room air (21%) and hypoxia (0.5%). A novel regulatory role of oxygen concentration on proliferation, senescence, and migration of CSCs is demonstrated that has implications for the production and development of CSC therapies.

Abstract

Cardiac stem cells (CSCs) are being evaluated for their efficacy in the treatment of heart failure. However, numerous factors impair the exogenously delivered cells' regenerative capabilities. Hypoxia is one stress that contributes to inadequate tissue repair. Here, we tested the hypothesis that hypoxia impairs cell proliferation, survival, and migration of human CSCs relative to physiological and room air oxygen concentrations. Human endomyocardial biopsy-derived CSCs were isolated, selected for c-Kit expression, and expanded in vitro at room air (21% O₂). To assess the effect on proliferation, survival, and migration, CSCs were transferred to physiological (5%) or hypoxic (0.5%) O₂ concentrations. Physiological O₂ levels increased proliferation (P < 0.05) but did not affect survival of CSCs. Although similar growth rates were observed in room air and hypoxia, a significant reduction of β-galactosidase activity (−4,203 fluorescent units, P < 0.05), p16 protein expression (0.58-fold, P < 0.001), and mitochondrial content (0.18-fold, P < 0.001) in hypoxia suggests that transition from high (21%) to low (0.5%) O₂ reduces senescence and promotes quiescence. Furthermore, physiological O₂ levels increased migration (P < 0.05) compared with room air and hypoxia, and treatment with mesenchymal stem cell-conditioned media rescued CSC migration under hypoxia to levels comparable to physiological O₂ migration (2-fold, P < 0.05 relative to CSC media control). Our finding that physiological O₂ concentration is optimal for in vitro parameters of CSC biology suggests that standard room air may diminish cell regenerative potential. This study provides novel insights into the modulatory effects of O₂ concentration on CSC biology and has important implications for refining stem cell therapies.

NEW & NOTEWORTHY

Physiological (5%) oxygen is optimal for proliferation and migration of c-kit⁺ cardiac stem cells (CSCs) compared with room air (21%) and hypoxia (0.5%). A novel regulatory role of oxygen concentration on proliferation, senescence, and migration of CSCs is demonstrated that has implications for the production and development of CSC therapies.

the efficacy of stem cell therapy for myocardial regeneration in heart failure is being actively investigated. Clinical trials have tested the use of c-Kit⁺ cardiac stem cells (CSCs) (6, 7) and bone marrow-derived mesenchymal stem cells (MSCs) (25, 29, 40) to reverse cardiac remodeling, reduce scar size, and improve cardiac and vascular function (5, 31, 33, 44). CSCs in particular, when transplanted into patients with ischemic cardiomyopathy, promoted enhancement in cardiac function, increased viable tissue, and improved measures of quality of life (17). Although these efficacy results are promising, numerous factors hamper the salutary effects of stem cell therapy, including cell survival, proliferation, and tissue engraftment (30, 45). Optimizing the therapeutic potential of delivered CSCs is essential to provide sustainable myocardial structural and functional improvements in patients with heart failure.

CSCs are a population of multipotent stem cells expressing CD117 (c-Kit) that reside in stem cell niches in the heart and have the capacity to differentiate into cardiomyocytes, endothelial cells, and cardiac conductive cells (5, 28, 46). However, a number of environmental and intrinsic stressors, including aging, replicative senescence, and disease, diminish CSC's regenerative and therapeutic effects (4, 35). Thus, strategies are being tested that enhance the survival, proliferation, and differentiation of CSCs (12, 23). Yet, none of these strategies fully address the cellular effects that result from the transfer of an in vitro expanded cell product to an in vivo postmyocardial injury microenvironment. In clinical trials, stem cells undergo isolation and ex vivo expansion in current good manufacturing practice (cGMP) facilities under a standard 21% oxygen (O₂)-rich environment, followed by intracoronary or transendocardial delivery into the heart. Once transplanted, stem cells face a shift toward O₂ concentrations that instantly drop to 5% in healthy myocardium and as low as 0–1% in an ischemic site (47). To date, the culture of stem cells in an artificial hyperoxic (21%) O₂ concentration is known to impair stemness and cell quality (22, 24). However, not much is known about how O₂ concentration regulates CSC regeneration and the precise molecular mechanisms governing the ability of CSCs to survive, proliferate, and migrate in a physiological or hypoxic myocardial microenvironment.

It has been previously shown that the development of hypoxic niches within the murine heart promotes the occurrence of a quiescent CSC phenotype (48). Interestingly, this effect decreased the amount of proliferating and senescent CSCs without altering mitochondrial volume, a major player in stem cell fate regulation (52). Additionally, numerous studies have focused on the survival of transplanted cells and the regulation of apoptotic cells after exposure to reactive oxygen and free radical stress (1). However, the sensitivity of CSCs to a combination of hypoxic- and free radical-induced apoptosis has not yet been tested. Last, endogenous CSC migration in myocardial infarction has been shown to be regulated by a number of O₂-sensitive signaling pathways, such as stromal cell-derived factor-1 (SDF-1) and C-X-C chemokine receptor type 4 (CXCR4) (36). The optimal O₂ concentration and impact of hypoxia on migration of exogenously delivered CSC has yet to be investigated.

We tested the hypothesis that hypoxic (0.5%) O₂ concentration impairs proliferation, survival, and migration of human CSCs compared with physiological (5%) and room air (21%) O₂ concentrations. The findings provide insight into the regulation of stem cell properties critical to successful cell therapy. Together, these results reveal basic mechanisms that affect aspects of human CSC biology in low O₂ environments, leading to a potential improvement in delivery strategies and/or cell production methods.

METHODS

Human CSC Isolation and Cell Culture

Human c-Kit⁺ CSCs were isolated from endomyocardial biopsies taken from patients, as previously reported (6, 18, 40). In brief, biopsies were plated on standard plastic cell culture plates until confluent cell expansion was achieved. After two cell passages, outgrown cells were selected based on positive expression of c-Kit using the Myltenyi magnetic sorting kit and c-Kit-APC conjugated antibody (ebiosciences). Biopsies and cells were maintained in Ham's F-12 media (GIBCO) containing 10% FBS, 10 ng/ml FGF, 5 mU/ml EPO, 0.2 mM glutathione, and 1% penicillin-streptomycin. Cells from three to four donors were used in all experiments for up to 10 passages. Various O₂ conditions were maintained using the thermo Scientific Heracell 150i incubator at 90% N₂-5% CO₂, 37°C for physiological O₂ concentration (5% O₂) and the Coy Laboratory hypoxia chamber at 94.5% N₂-5% CO₂, 37°C for hypoxic O₂ levels (0.5% O₂).

Flow Cytometry Analysis

CSCs in culture were trypsinized and washed with fluorescence-activated cell sorting (FACS) buffer before staining with CD45 (555482; BD Pharmingen) and CD117 (17–1171; eBioscience) antibodies. After surface antibody staining, cells were washed again with FACS buffer and fixed with cytofix/cytoperm buffer (BD Biosciences). Cells were then incubated again with CD117 antibody to ensure staining of total cell surface and internalized CD117 expression. After this final antibody incubation, cells were washed with FACS buffer and fixed with 2% paraformaldehyde. Data were collected using BD Aria II LSRII flow cytometry (Bio-Rad) and analyzed with FloJo software in the Sylvester Comprehensive Cancer Center Flow Cytometry Core Facility.

Western Blotting

Protein extracts were quantified by standard Bradford assay to ensure equivalent protein loading onto Criterion Tris·HCl gels (Bio-Rad) and transferred to a nitrocellulose membrane. Membranes were blocked with 5% milk in TBST (1× Tris-buffered saline-0.1% Tween) for 1 h followed by overnight incubation with the following primary antibodies: sirtuin 1 (SIRT1, 1:2,000; Abcam ab110304), P16 (1:1,000; Abcam ab108349), and actin (1:1,000; Santa Cruz sc-1616). The blots were then washed three times with TBST buffer followed by incubation with horseradish peroxidase-conjugated secondary antibody. Blots were imaged using Super Signal Chemiluminescent substrate (Thermo) and the Chemi Doc XRS imagining system (Bio-Rad).

Gene Expression Analysis

Total RNA was isolated from cell and tissue lysates using the RNeasy mini plus kit (Qiagen) according to the manufacturer's instructions. RNA was then converted to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems) before analysis by quantitative PCR. Quantitative PCR reactions were done using Taqman Universal Master Mix (Applied Biosystems) and Taqman gene expression probes (Thermo) in an iQ5 real-time PCR detection system (Bio-Rad). Samples were run in duplicate and normalized to the housekeeping gene GAPDH. Gene expression changes were calculated using the 2−ΔΔC_t method. The probes used for gene expression analysis are as follows: SDF-1 (CXCL12, human) Hs03676656_mH, CXCR4 (human) Hs00607978_s1, SDF-1 (CXCL12, mouse) Mm00445553_m1, and CXCR4 (mouse) Mm01996749_s1.

Proliferation Assays

Human CSCs were seeded at a density of 1.0 × 10⁵ on 100 × 20 mm cell culture dishes and counted with the TC10 automated cell counter (Bio-Rad) after 24, 72, and 96 h of growth in various oxygen conditions. Doubling rates were then calculated using the equation:

$$[96\hspace{0.17em}\text{h}\hspace{0.17em}\times \hspace{0.17em}\log \hspace{0.17em}(2)]/[\log \hspace{0.17em}(\text{end}\hspace{0.17em}\text{point})-\log \hspace{0.17em}(start\hspace{0.17em}point)].$$

Lentivirus Transduction

Human SIRT1 p-GIPZ silencing plasmid (Dharmacon) and pDsRed2-mito vector (Clontech) were transfected, along with lentiviral packaging plasmids VSVG and Gagpol (Addgene), into 293T cells using Lipofectamine 2000 (Life Technologies). The pLV-CMV-MT-dsRED-Puro was created by PCR amplifying the Mito-dsRED fragment using the pDsRed2-Mito plasmid (Clontech) as template; then, the MT-dsRED fragment was subcloned in place of the GFP fragment into the pLenti-CMV-GFP-Puro plasmid (Addgene 17448) at XbaI and SalI sites. Lentiviral particles were concentrated through precipitation from culture supernatants using PEG-IT (System Biosciences). Lentivirus concentration was then quantified with the QuickTiter Lentivirus Titer Kit (Cell Bio Labs). CSCs were seeded 24 h before being transduced at a ratio of 1.0 × 10¹⁰ lipopolysaccharide/2.00 × 10⁵ cells/100 mm dish for 5 h in serum-free F-12 medium. After this time, complete media was added. Efficiency of transduction was determined by identification of green fluorescent protein (GFP) or DsRed-positive cells by fluorescence microscopy. Cells carrying viral particles were selected by addition of puromycin.

β-Galactosidase Senescence Assay

Replicative senescence was determined by the fluorometric cellular senescence-associated β-galactosidase (β-Gal) activity assay (Cell Bio Labs). Cells were harvested after 24 and 96 h of growth, and protein lysates were prepared according to the manufacturer's instructions. Sample concentration was determined by a standard Bradford assay, and 20 μg of protein were used to detect β-Gal activity in a reaction of 3 h. Fluorometric intensity was then measured using the Spectra Max M5 plate reader (Molecular Devices).

Glycolysis assay.

The measurement of cellular glycolysis was performed using the Glycolysis Cell-Based Assay Kit (Cayman Chemical) according to the manufacturer's instructions. In short, CSCs were grown in various O₂ concentrations for 96 h, collected, and replated at a density of 1 × 10⁴ cells/well in a 96-well plate. After cell attachment, culture media was replaced with F-12 media containing 1% FBS for overnight incubation. Supernatants were collected, and the concentration of l-lactate was determined by measuring absorbance of reaction samples using the Spectra Max M5 plate reader (Molecular Devices).

Apoptosis Detection

Apoptotic and necrotic cells were identified by live staining with annexin V, Pacific Blue conjugate (Life Technologies) and propidium iodide (Life Technologies). Briefly, 1.0 × 10⁵ cells were plated on 60 × 15 mm cell culture dishes and incubated in serum-free F-12 media overnight in various O₂ conditions. Apoptosis was induced by treatment with 500 μM H₂O₂. After 3 h, cells were trypsinized, washed two times with phosphate-buffered saline, and stained with 5 μl of annexin V conjugate and 1 μg/ml propidium iodine in 1X binding buffer (Life Technologies). Data were collected using BD Aria II LSRII flow cytometry (Bio-Rad) and analyzed with FloJo software.

Transwell Migration

The ability of CSCs to migrate was determined by using 0.8-μM-pore size transwell inserts (Falcon) on 24-well plates. Human MSCs and CSCs were seeded at a density of 1 × 10⁵ cells on a 24-well plate overnight followed by media replacement with starvation media (serum-free F-12 media containing 0.1% BSA). Starvation media alone was used as the control. Transwells with 100 μl of starvation media containing 1 × 10⁵ CSCs were placed to the prepared wells, and migration was allowed for 24 h. After completion of the assay, the transwells were removed and cleaned of excess media and cells with a cotton-tipped applicator. The bottoms of the transwells were then fixed with 70% ethanol and stained with 0.2% crystal violet for 5–10 min. Stained membranes were then washed and allowed to dry before imagining on a Nikon Eclipse light microscope. A minimum of three pictures was taken of different fields within each transwell replicate at ×4 magnification using a Nikon Digital Sight camera. Cell migration was determined by counting the number of crystal violet-stained cells on the underside of the transwell apparatus. The level of cell migration under starvation media in room air served as the standard control for all experiments.

c-Kit-IRG transgenic Mice and Migration Assay

The cKit^CreERT2/+, IRG, Isl1-nLacZ, Wnt1-Cre, tdTomato, Wnt1::Flpe, and RC::Fela mice that were employed in this study have been described previously (28). To induce CreER^T2-mediated recombination and specific GFP labeling of c-Kit⁺ cells, tamoxifen (Sigma) was prepared by resuspension in peanut oil (Sigma) at a final concentration of 20 mg/ml by shaking overnight at 37°C. Tamoxifen was then administered via single subcutaneous injections (50 μl/injection) to 2- to 4-day-old neonatal pups. One day postinjections, hearts were harvested, washed in ice-cold HBSS, and cleared of unwanted tissues under a stereomicroscope (VistaVision). These hearts were further processed in a tissue-cultured hood following additional washing steps with DMEM (GIBCO). Heart explants were created by mincing neonatal hearts into ∼2- to 3-mm³ fragments and digested in a solution of DMEM-F-12 (GIBCO), 20% FBS, 1% penicillin/streptomycin, and 200 U/ml collagenase type II solution (Worthington) at 37°C. Following two washes with DMEM to remove residual enzyme, digested explants were hand-picked under sterile conditions with a micropipette and cultured individually on gelatin-coated 24-well plates. Samples were fed every other day for the 1-wk culture period with medium consisting of DMEM-F-12, 15% FBS (Atlas), 1% penicillin/streptomycin (GIBCO), 1% β-mercaptoethanol (GIBCO), 1,000 U/ml recombinant mouse LIF (Millipore), 1 ng/ml recombinant mouse bFGF (Peprotech), 100 ng/ml recombinant murine SCF (Peprotech), and 0.1 mM nonessential amino acids (GIBCO). The number of GFP⁺ cells was counted from pictures taken after 1 wk of culture based on GFP epifluorescence, under a fluorescent microscope (Olympus IX81).

Statistical Analysis

The results are expressed as means ± SE. A P value <0.05 was considered significant. Differences between groups were examined for statistical significance using Student's t-test or analysis of variance (ANOVA), with Newman-Keuls multiple-comparisons test where appropriate for post hoc analyses. Differences within groups are examined by two-way ANOVA and the Newman-Keuls multiple-comparison post hoc test. Experiments were repeated a minimum of three separate times using cells from three or more different donors.

RESULTS

CSC Proliferation is Enhanced in Physiological Oxygen Concentration

CSC isolation was performed by the University of Miami cGMP facility, according to established protocols (6, 18, 40). Endomyocardial biopsies were obtained from patients enrolled in the POSEIDON-DCM (40) clinical trial. CSCs were isolated and expanded from the biopsies and magnetically selected by c-Kit (CD117) positivity. Characterization by flow cytometry confirmed the selection of CD117⁺ and CD45⁻ CSCs (Fig. 1, A–C). Purified cultures from various donors were further expanded in culture and used in all experiments (Fig. 1D).

Fig. 1.

Characterization of human cardiac stem cells (CSCs). A and B: flow cytometry analysis of cardiac-derived CD117 (c-Kit)-positive and CD45-negative cells confirms a pure selection of CSCs. C: table representing the c-Kit positivity of cell populations isolated from three separate donors. D: representative image (×4 magnification) showing cell morphology of expanded CSC.

To elucidate the effect of O₂ transition on CSC proliferation, a standard growth curve analysis of 0.5 × 10⁵ cells was performed for 96 h. CSCs grown at room air were transferred to either physiological (5%) or hypoxic (0.5%) O₂ concentrations. The recorded cell counts demonstrated increased cell proliferation under physiological O₂ conditions at 72 h compared with room air and hypoxia (4.4 ± 0.4 × 10⁵ cells in physiological, 3.1 ± 0.3 × 10⁵ cells in room, and 2.8 ± 0.2 × 10⁵ cells in hypoxia, P < 0.01). Cell proliferation continued to increase at physiological O₂ at 96 h compared with room air or hypoxia (7.6 ± 0.6 × 10⁵ cells in physiological, 5.0 ± 0.3 × 10⁵ cells in room, and 4.3 ± 0.4 × 10⁵ cell in hypoxia, P < 0.001) (Fig. 2A). However, there was no significant difference in proliferation when CSCs were grown in room air or hypoxia. The calculated doubling time of CSCs gown in physiological O₂ decreased relative to both room air and hypoxic O₂ concentrations (23.4 ± 0.7 h in physiological, 27.6 ± 1 h in room, and 29.1 ± 1.2 h in hypoxia, P < 0.05) (Fig. 2B).

Fig. 2.

Effect of oxygen concentration on CSC proliferation. A: growth curve analysis shows proliferation of CSCs grown under room, physiological, and hypoxic O₂ concentrations (n = 8). B: the doubling time of CSCs grown under various O₂ concentrations (n = 8). C: Western blot analysis of sirtuin 1 (SIRT1, n = 13), with calculated fold change relative to room O₂ concentration after 96 h of growth. Actin is used as a protein loading control. D: growth curve analysis of CSCs transduced with SIRT1 knockdown (KD) small interfering RNA (siRNA) (n = 3). E: the doubling time of SIRT1 KD and scrambled siRNA control CSCs. F: Western blot for SIRT1 confirms successful siRNA silencing. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

SIRT1 is a regulator of cell proliferation that plays a key role in cellular responses to environmental stressors (9, 42, 51, 54). To test the involvement of SIRT1 in the O₂-mediated effect on cell proliferation, SIRT1 protein expression was measured at the 96-h time point. Consistent with the growth curve data, SIRT1 was upregulated in CSCs cultured at physiological O₂ compared with room air (1.375 ± 0.9-fold, P < 0.01) O₂ concentration (Fig. 2C). There was no change in SIRT1 expression after growth in hypoxia relative to room O₂ (0.85 ± 0.9-fold, P > 0.05). The requirement of SIRT1 in CSC proliferation was confirmed by lentiviral transduction of specific small interfering RNA gene knockdown (KD) under room air conditions. SIRT1 KD significantly decreased the number of CSCs grown over 96 h (4.35 ± 0.3 × 10⁵ scrambled control cells and 1.75 ± 0.2 × 10⁵ SIRT1 KD cells, P < 0.0001) and increased the doubling time (23.3 ± 0.3 h for scrambled control and 34.17 ± 0.3 h for SIRT1 KD, P < 0.0001) (Fig. 2, D–F).

Hypoxia Decreases Senescence and Mitochondrial Content and Increases Glycolysis

To determine whether the decreased CSC proliferation observed in hypoxia and room air, relative to physiological O₂, was due to senescence, we tested the expression of the senescence-associated markers β-Gal and P16INK4a. Detection of β-Gal activity in cell lysates decreased within 96 h of growth in both physiological and hypoxic O₂ concentrations but remained unchanged at room air (−2,924 ± 984 fluorescent units in physiological, P < 0.05, −4,203 ± 1,065 fluorescent units in hypoxia, P < 0.05, and −139 ± 155 fluorescent units in room air, P > 0.05; Fig. 3A). Additionally, the level of β-Gal activity at 96 h relative to room air was significantly lower only in the hypoxia-treated group (12,272 ± 727 fluorescent units in hypoxia and 15,502 ± 841 fluorescent units in room air, P < 0.05; Fig. 3A). Similarly, P16INK4a expression was downregulated after 96 h of growth in hypoxia (0.58 ± 0.1-fold, P < 0.001) but not in physiological oxygen (0.96 ± 0.02-fold, P > 0.05; Fig. 3B) compared with room air. These findings suggest that the impaired cell proliferation effect observed in room and hypoxic O₂ concentration, relative to physiological O₂ concentration, is under the regulation of different mechanisms.

Fig. 3.

Replicative senescence and mitochondrial content in human CSCs. A: senescence-associated β-galactosidase (β-Gal) activity after 24 and 96 h of O₂ exposure was determined by the reactivity with a fluorescent substrate (n = 5). B: Western blot analysis of P16 (n = 6) with calculated fold change relative to room air treatment after 96 h. Actin is used as a protein loading control. C: fluorescent images of CSCs transduced with pDSRed2-Mito vector after transfer to room air, physiological, and hypoxic O₂ for 96 h (n = 3). D: flow cytometry analysis of the mean fluorescent intensity of pDSRed2-Mito transduced CSCs after 24 and 96 h of various O₂ exposures. E: the calculated fold change in DSRed2 mean fluorescent intensity relative to respective room air control. F: the concentration of l-lactate detected in supernatant of CSCs grown in various O₂ concentrations for 96 h (n = 6). Data are presented as means ± SE. ⁺Two-way ANOVA, P < 0.05 (within-group comparison). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Mitochondria are key regulators of stem cell fate commitment, stem cell maintenance, and redox balance (11, 16, 37). We tested the effect of different O₂ concentrations on CSC mitochondrial content by transducing CSCs with a lentiviral pDSRed-Mito vector for specific and prolonged fluorescent labeling of the mitochondria (Fig. 3C). Alterations in total mitochondrial content after 24 and 96 h of exposure to physiological and hypoxic O₂ were assessed by flow cytometric analysis of the DsRed mean fluorescent intensity. We found that hypoxic O₂ reduced the total MFI of mitochondria after 24 h (0.75 ± 0.1-fold, P < 0.05), with the greatest reduction at 96 h (0.18 ± 0.03-fold, P < 0.0001) compared with the room O₂ group. The transfer from room air to physiological O₂ did not significantly decrease mitochondrial MFI after 24 h (0.94 ± 0.05-fold, P > 0.05) but did have a decreasing effect over the 96-h time course (0.6 ± 0.2-fold, P < 0.05) (Fig. 3, D and E). Together these results confirm a role of oxygen in the maintenance and abundance of total mitochondria.

CSC anerobic metabolism was also measured by recording glycolytic activity via supernatant l-lactate detection. The concentration of supernatant l-lactate was elevated after growth in both physiological (1.23 ± 0.08 mM, P < 0.01) and hypoxic (1.13 ± 0.09 mM, P < 0.05) O₂ concentrations relative to room air (0.9 ± 0.05 mM), demonstrating the expected increase in glycolysis activity (Fig. 3F) and metabolic adaptation to decreased O₂ concentrations.

CSC Sensitivity to Oxidative Stress is Similar in All O₂ Concentrations

Cell survival posttransplantation may be the most critical contributor to successful cell therapy. To test the impact of O₂ concentration on cell sensitivity to apoptosis, CSCs from various donors were cultured in room air, physiological, and hypoxic O₂ concentrations, stimulated with the proapoptotic agent H₂O₂, and analyzed for the induction of apoptosis via positive annexin V staining. Flow cytometry analysis recorded the percentage of early apoptotic cells by gating cells based on annexin V positivity and propidium iodine negativity (Fig. 4A). Late apoptotic or necrotic cells were identified as annexin V-positive and propidium iodine-positive cells. A similar percentage of apoptotic CSCs resulted in all O₂ concentrations, relative to the full media control, 3 h post-H₂O₂ stress (4.9 ± 0.8% in full media control, 15.89 ± 0.9% in room O₂, 16.24 ± 1.1% in physiological O₂, and 20.82 ± 2.9% in hypoxic O₂) (Fig. 4B). Additionally, O₂ concentration did not influence the H₂O₂-induced necrosis (3.58 ± 0.7% in full media control, 11.49 ± 2.3% in room O₂, 13.34 ± 2.7% in physiological O₂, and 11.78 ± 2.5% in hypoxic O₂) (Fig. 4C).

Fig. 4.

Effect of oxygen on human CSC apoptosis and necrosis. A: flow cytometry analysis of annexin V- and propidium iodine (PI)-stained CSCs 3 h post-H₂O₂ stress in various O₂ concentrations. B: the percentage of early apoptotic (annexin V⁺/PI⁻) in all treatments (n = 3). C: the percentage of late apoptotic/necrotic (annexin V⁺/PI⁺) in all treatments (n = 3). Data are presented as means ± SE.

CSC Migration is Enhanced in Physiological O₂

To study the effect of O₂ concentrations on CSC migration, a transwell migration assay was used. Positive cell migration was recorded by counting crystal violet-stained cells on the underside of the transwell apparatus. Under starvation media conditions, there was a basal level of CSC migration within 24 h that became enhanced under physiological O₂ conditions (2.40 ± 0.13-fold, P < 0.0001) and unchanged in hypoxia (1.11 ± 0.15-fold, P > 0.05) relative to room O₂ concentration (Fig. 5, A and B). Bone marrow-derived MSCs promote chemotactic and proliferative responses in CSC via the SDF1/CXCR4 pathway (27). Therefore, to investigate the potential use of MSCs to enhance CSC migration under hypoxic and room air conditions, CSCs were exposed to stimulus from human MSCs or CSCs under similar starvation media conditions. Upon addition of the human MSCs, the migration of CSCs was enhanced in hypoxic O₂ to a level comparable to physiological migration (Fig. 5, A and C). A similar trend for MSC-improved migration was found in room air. There was no effect found with the addition of CSCs. RNA analysis of promigratory genes showed that the transfer of CSCs to hypoxia caused a significant reduction in both SDF-1 (0.57 ± 0.09-fold, P < 0.01) and CXCR4 (0.52 ± 0.12-fold, P < 0.05) gene transcripts relative to room air O₂. However, there was no significant difference in SDF-1 (0.99 ± 0.11-fold, P > 0.05) or CXCR4 (0.71 ± 0.14-fold, P > 0.05) expression in CSCs in the physiological O₂ group relative to room air O₂ (Fig. 5, D and E).

Fig. 5.

Effect of oxygen on CSC migration. A: representative images of transwell-migrated CSCs stained with crystal violet under various treatment conditions (n = 4). Positive crystal violet staining indicates migratory cells. B and C: migratory CSCs were counted, and the value of CSC migration is displayed as the fold change relative to the room air control. D and E: RNA expression of stromal cell-derived factor-1 (SDF-1) and C-X-C chemokine receptor type 4 (CXCR4) represented as fold change relative to the room air control after normalization to the housekeeping gene GAPDH (n = 6). Data are presented as means ± SE. *P < 0.05, **P < 0.01, and ****P < 0.0001.

These findings were further confirmed via the analysis of c-Kit⁺/GFP⁺ CSC migration from neonatal murine cardiac explants. The murine model used ensures specific and accurate GFP labeling of c-Kit⁺ cells within the tissue after tamoxifen treatment. Migration of GFP⁺ cells was then monitored over a 1-wk culture period, and a significantly elevated c-Kit⁺ CSC migration was observed in cultures grown in physiological O₂ (20.0 ± 1.3 cells, P < 0.05) compared with room air (2.22 ± 1.3 cells) and hypoxic O₂ (5.33 ± 2.92 cells) (Fig. 6, A and B). Gene expression analysis also showed a decrease in SDF-1 mRNA in hypoxic samples (0.47 ± 0.09-fold, P < 0.001) relative to room air but no significant difference in CXCR4 expression (1.3 ± 0.45-fold, P > 0.05) (Fig. 6, C and D). Gene expression of SDF-1 and CXCR4 was also similar between room air and physiological O₂ groups (1.10 ± 0.13- and 1.81 ± 0.50-fold, respectively, P > 0.05).

Fig. 6.

Oxygen-dependent effect of CSC migration from neonatal murine explants. A: representative images of migrated green fluorescent protein-positive (GFP⁺)/c-Kit⁺ CSCs from neonatal murine explants after a 1-wk culture in various O₂ concentrations (n = 3). B: the total number of migratory cells in each O₂ concentration. C and D: RNA expression of SDF-1 and CXCR4 represented as fold change relative to the room air control after normalization to the housekeeping gene GAPDH (n = 9). Data are presented as means ± SE. *P < 0.05, ***P < 0.001, and ****P < 0.0001.

DISCUSSION

The primary findings of this study demonstrate a major role of O₂ concentration in the regulation of human CSC biology and mechanisms of proliferation and cell migration. In stem cell-based therapies, cell products are first isolated and expanded from tissues in a hyperoxic (21% O₂) in vitro culture environment before transplantation back into tissues (55). Although donor age, patient health status, and feasibility of cell maintenance in culture are known to influence the potency of delivered cells, minimal information has been reported concerning the effect of lower O₂ concentrations on human CSC regenerative potential (13, 15, 20). The objective of this study was to identify basic in vitro effects of O₂ concentration on parameters of CSC biology that impact the therapeutic effectiveness of stem cell therapy. Thus, our result that the transfer of human CSCs to physiological (5%) O₂ provides superior benefit to their proliferative and migratory ability provides novel insight into potential improvements in cell preparation techniques and regulatory mechanisms that affect survival and integration posttransplantation in the injured myocardium.

O₂ has been a well-established regulator of stem cell proliferation from within and outside the stem cell niche (2, 38). Typically, hypoxic stem cell niches promote reversible cell cycle arrest or quiescence while maintaining a nondifferentiated state (21). However, to what degree hypoxia has an effect on cell proliferation seems to vary based on cell type and origin (2). In general, stem cells prepared for therapy are expanded at a level of O₂ far greater than that of the physiological tissue. Thus, transplantation introduces various degrees of hypoxic stress depending on tissue viability and perfusion. Therefore, we tested the effect of cell transition to two different degrees of hypoxia relative to standard laboratory room air that are comparable to a physiological and ischemic/hypoxic environment (47).

Our initial findings show an enhanced proliferation rate of those CSCs transferred to physiological vs. hypoxic O₂. However, no significant difference was found comparing CSCs grown at room air vs. hypoxia. Interestingly, there appears to be a “sweet spot” in O₂ concentration gradient that favors cell expansion. This finding is reflective of the concept that the expansions of CSCs in an unnatural hyperoxic or hypoxic condition are equally stressful. We then tested the expression level of the cell cycle regulator SIRT1. Acting as a NAD⁺-dependent histone deacetylase, SIRT1 is a well-established target for cellular “longevity” through its role in cellular adaptation to stress and protection against nonreversible cell cycle arrest or senescence (3, 8). SIRT1 confers protection against cell cycle arrest by promoting the inhibition of cell cycle inhibitor proteins P53 and retinoblastoma (51, 54). Accordingly, we found that SIRT1 is necessary for the preservation of CSC proliferation and that SIRT1 protein expression is regulated by O₂ concentration. In fact, the upregulation of SIRT1 in physiological O₂ suggests a potential mechanism that favors optimal cell proliferation at this O₂ concentration.

SIRT1 protein expression promotes protection against senescence (10, 42). Therefore, we tested the expression of senescence-associated markers in CSCs exposed to physiological and hypoxic O₂ concentrations compared with room air. Similar to those results reported by Sanada et al., we found that CSCs exposed to hypoxic (0.5%) O₂ had a reduced cellular senescence phenotype, measured by a decreased expression in β-Gal activity and P16INK4a protein (48). Notably, although cell proliferation rate was found to be similarly decreased in room air and hypoxia relative to physiological O₂, the differences in senescence marker expression suggest that O₂ concentration regulates CSC growth through different pathways. The elevated levels of β-Gal activity and P16INK4a in room O₂ indicate a greater population of replicative senescence, whereas a decrease in these markers suggests accumulation of a quiescent phenotype in hypoxia.

Further evidence that growth in hypoxia leads to a quiescent phenotype is shown by the measurement of mitochondrial content and glycolytic activity. As the respiration centers of the cell, an increase in mitochondrial biogenesis and elevated rate of oxygen consumption has been linked to stem cell differentiation, reprograming, and replicative senescence. On the other hand, decreased levels correlate with increased stemness and self-renewal (41, 52). Specifically, MSCs grown at room air O₂ concentration have compromised colony-formation capacity through the promotion of reactive oxygen species-induced senescence (39, 43). Thus, our observation that hypoxic O₂ concentration promotes decreased mitochondrial content, elevated glycolysis, and decreased senescence-associated markers in CSCs, relative to the room air and physiological oxygen groups, provides strong evidence of hypoxia-induced quiescence. This finding concurs with previously published data concerning hypoxic regulation of stem cell biology. The reduction in mitochondrial respiration and switch to glycolysis in CSCs may serve as an adaptive mechanism that allows the preservation of a slow growth state, free of oxidative stress damage, and ultimately reducing replicative senescence.

An immediate obstacle that CSCs transplanted in the heart must overcome is survival from oxidative damage (30, 32). The ability of CSCs to resist cell death is a critical component of stem cell therapy that needs to be improved (1). It has been reported by Hong et al. that the vast majority of intracoronary infused CSCs do not survive in the heart longer than 24 h (30). Therefore, we tested the short-term effect of transfer to lower O₂ concentration on cell apoptosis and survival. CSCs were transferred to physiological and hypoxic O₂ concentration and treated with the proapoptotic agent H₂O₂. Although there was not a consistent effect of O₂ on H₂O₂-induced apoptosis and necrosis, those CSCs transferred to hypoxia did have a trend that suggests a possible increase in sensitivity to apoptotic stress.

CSCs need to migrate and integrate in the tissue to sufficiently contribute to the repair of the injured myocardium (36). As expected, we found an O₂-dependent regulation of CSC migration. Similar to the effect on cell proliferation, we found physiological O₂ concentration enhanced CSC migration relative to both room air and hypoxic O₂ concentrations. Importantly, CSC migration and proliferation is influenced by the secretion and expression of the promigratory proteins SDF-1 and CXCR4 (14, 27). The similar expression levels of SDF-1 and CXCR4 mRNA in CSCs exposed to room air and physiological O₂, however, do not offer an explanation for enhanced migration. Rather, it can be hypothesized that similar pathways, perhaps related to senescence, regulate physiological oxygen's positive effect on proliferation and migration relative to room air (19). On the other hand, the placement of CSCs in hypoxia did reduce the transcription levels of SDF-1 and CXCR4, suggesting a potential mechanism underlying the impaired migration at this O₂ concentration.

The finding that physiological O₂ concentration enhances CSC migration was further confirmed using a murine model that utilized the accurate and specific labeling of c-Kit⁺ CSCs with genetic GFP recombination. With this model, migrating CSCs from cardiac explants are tracked in cell culture. We found a greater number of migratory CSCs moving out of neonatal mouse cardiac explants in physiological O₂ relative to both hypoxia and room air. This finding has direct implications on the process in which CSCs are isolated and expanded from patient endomyocardial biopsies in vitro. CSCs are a small population of cells within the heart, making it a challenge to isolate and expand a large number of cells in a short period of time before transplantation. Our data indicate that culturing endomyocardial explants in physiological O₂ improves this challenge.

Although our findings contrast with that of others who found that hypoxic preconditioning enhances murine CSC migration and tissue integration, there are important differences in study methodology that may account for this discrepancy (49, 50, 56). In general, studies have reported that short-term activation of HIF-1α-regulated targets by hypoxic preconditioning activates numerous prosurvival and promigratory pathways that enhance stem cell function upon transplantation. In our model, we test the migratory ability of standard room air-cultured CSCs over 24 h of exposure to physiological or hypoxic O₂. We do find that reducing O₂ concentration below that of room air enhances CSC migration; however, this benefit is lost with the extreme low levels that are considered hypoxia (0.5% O₂). We believe that these differences may be due to the studied cell type. Human CSCs isolated from patients' endomyocardial biopsies introduce several variability factors, such as cell source conditions and patient comorbidities. These conditions, as well as species differences, may account for the decreased migratory function in extreme hypoxic stress that this study found in human CSCs compared with studies with murine-derived CSCs.

We further sought to rescue the defect in migration of CSCs by introducing conditioned MSC media. MSCs secrete many soluble factors, including SDF-1, and positively regulate CSC proliferation and migration (26, 27, 57). Our result was consistent, indicating that MSCs promote CSC migration under hypoxic conditions. This finding further strengthens the argument that a combination of CSCs with MSCs may provide superior benefit in cell regeneration (34, 53).

In summary, our results show that O₂ concentration modulates CSC proliferation and migration. These findings have important implications for cell manufacturing and further validate the transplantation of CSCs in the viable border zone of an ischemic injury, where O₂ concentration is at a physiological level. The characterization of a quiescent phenotype upon transfer to a hypoxic environment may in part explain a blunted CSC-mediated regenerative response. We have previously shown that coadministration of CSCs with MSCs provides superior benefit to CSC cell function and overall cardiac repair (26, 53). Consistent with those findings, we show here that MSCs promote the migration of CSCs in hypoxia, further supporting the notion that MSCs may serve as a viable option for reducing hypoxic-induced quiescence and promoting CSC regeneration. Additionally, CSCs are being currently isolated and expanded in room air culture conditions before cell transplantation. Our finding that physiological O₂ concentration reduces CSC senescence and enhances migratory ability suggests that the isolation and expansion of CSCs at physiological O₂ concentration may offer a better therapeutic option for cell preparation. Finally, the identification of various cell biology parameters and pathways that are regulated by changes in O₂ concentration serve as a starting point for further exploration and techniques that could improve CSC regenerative ability in hypoxic tissues.

GRANTS

This work was supported by an American Heart Association predoctoral award (M. A. Bellio), National Heart, Lung, and Blood Institute Grants UM1-HL-113460, R01-HL-084275, R01-HL-107110, and R01-HL-110737, the Starr Foundation, and the Soffer Family Foundation.

DISCLOSURES

Dr. Hare has a patent for cardiac cell-based therapy; he holds equity in Vestion Inc.; maintains a professional relationship with Vestion as a consultant and member of the Board of Directors and Scientific Advisory Board; and is a shareholder in Longeveron LLC. The other authors declare that they have no competing interests.

AUTHOR CONTRIBUTIONS

M.A.B., J.M.H., and I.H.S. conception and design of research; M.A.B., K.E.H., V.F., K.V., J. K., and A.K. performed experiments; M.A.B., A.M.L., and K.E.H. analyzed data; M.A.B., C.O.R., A.M.L., K.E.H., V.F., J.M.H., and I.H.S. interpreted results of experiments; M.A.B. prepared figures; M.A.B. drafted manuscript; M.A.B., C.O.R., A.M.L., J.M.H., and I.H.S. edited and revised manuscript; M.A.B., C.O.R., A.M.L., K.E.H., V.F., K.V., A.K., J.M.H., and I.H.S. approved final version of manuscript.