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1.  Hypoxic Preconditioning Results in Increased Motility and Improved Therapeutic Potential of Human Mesenchymal Stem Cells 
Stem cells (Dayton, Ohio)  2008;26(8):2173-2182.
Mesenchymal stem cells (MSC) are adult multipotent cells found in bone marrow, adipose tissue, and other adult tissues. MSC have been shown to improve regeneration of injured tissues in vivo, but the mechanisms remain unclear. Typically, MSC are cultured under ambient, or normoxic, conditions (21% oxygen). However, the physiological niches for MSC in the bone marrow and other sites have much lower oxygen tension. When used as a therapeutic tool to repair tissue injuries, MSC cultured in standard conditions must adapt from 21% oxygen in culture to less than 1% oxygen in the ischemic tissue. We therefore examined the effects of preculturing human bone marrow-derived MSC in hypoxic conditions (1%–3% oxygen) to elucidate the best conditions that enhance their tissue regenerative potential. We demonstrated that MSC cultured in hypoxia activate the Akt signaling pathway while maintaining their viability and cell cycle rates. We also showed that MSC cultured in hypoxia induced expression of cMet, the major receptor for hepatocyte growth factor (HGF), and enhanced cMet signaling. MSC cultured in hypoxic conditions increased their migration rates. Since migration and HGF responsiveness are thought to be key mediators of MSC recruitment and/or activation in vivo, we next examined the tissue regenerative potential of MSC cultured under hypoxic conditions, using a murine hind limb ischemia model. We showed that local expression of HGF is increased in ischemic muscle in this model. Intra-arterial injection of MSC cultured in either normoxic or hypoxic conditions 24 hours after surgical induction of hind limb ischemia enhanced revascularization compared with saline controls. However, restoration of blood flow was observed significantly earlier in mice that had been injected with hypoxic preconditioned MSC. Collectively, these data suggest that preculturing MSC under hypoxic conditions prior to transplantation improves their tissue regenerative potential.
doi:10.1634/stemcells.2007-1104
PMCID: PMC3017477  PMID: 18511601
Immune-deficient mice; Human stem cells; Mesenchymal stem cells; Hypoxia; Transplantation; Tissue repair
2.  Hypoxia Promotes Osteogenesis but Suppresses Adipogenesis of Human Mesenchymal Stromal Cells in a Hypoxia-Inducible Factor-1 Dependent Manner 
PLoS ONE  2012;7(9):e46483.
Background
Bone fracture initiates a series of cellular and molecular events including the expression of hypoxia-inducible factor (HIF)-1. HIF-1 is known to facilitate recruitment and differentiation of multipotent human mesenchymal stromal cells (hMSC). Therefore, we analyzed the impact of hypoxia and HIF-1 on the competitive differentiation potential of hMSCs towards adipogenic and osteogenic lineages.
Methodology/Principal Findings
Bone marrow derived primary hMSCs cultured for 2 weeks either under normoxic (app. 18% O2) or hypoxic (less than 2% O2) conditions were analyzed for the expression of MSC surface markers and for expression of the genes HIF1A, VEGFA, LDHA, PGK1, and GLUT1. Using conditioned medium, adipogenic or osteogenic differentiation as verified by Oil-Red-O or von-Kossa staining was induced in hMSCs under either normoxic or hypoxic conditions. The expression of HIF1A and VEGFA was measured by qPCR. A knockdown of HIF-1α by lentiviral transduction was performed, and the ability of the transduced hMSCs to differentiate into adipogenic and osteogenic lineages was analyzed. Hypoxia induced HIF-1α and HIF-1 target gene expression, but did not alter MSC phenotype or surface marker expression. Hypoxia (i) suppressed adipogenesis and associated HIF1A and PPARG gene expression in hMSCs and (ii) enhanced osteogenesis and associated HIF1A and RUNX2 gene expression. shRNA-mediated knockdown of HIF-1α enhanced adipogenesis under both normoxia and hypoxia, and suppressed hypoxia-induced osteogenesis.
Conclusions/Significance
Hypoxia promotes osteogenesis but suppresses adipogenesis of human MSCs in a competitive and HIF-1-dependent manner. We therefore conclude that the effects of hypoxia are crucial for effective bone healing, which may potentially lead to the development of novel therapeutic approaches.
doi:10.1371/journal.pone.0046483
PMCID: PMC3459928  PMID: 23029528
3.  Increased Proliferation and Analysis of Differential Gene Expression in Human Wharton's Jelly-derived Mesenchymal Stromal Cells under Hypoxia 
Multipotent mesenchymal stromal cells (MSCs) from Wharton's jelly (WJ) of umbilical cord bear higher proliferation rate and self-renewal capacity than adult tissue-derived MSCs and are a primitive stromal cell population. Stem cell niche or physiological microenvironment plays a crucial role in maintenance of stem cell properties and oxygen concentration is an important component of the stem cell niche. Low oxygen tension or hypoxia is prevalent in the microenvironment of embryonic stem cells and many adult stem cells at early stages of development. Again, in vivo, MSCs are known to home specifically to hypoxic events following tissue injuries. Here we examined the effect of hypoxia on proliferation and in vitro differentiation potential of WJ-MSCs. Under hypoxia, WJ-MSCs exhibited improved proliferative potential while maintaining multi-lineage differentiation potential and surface marker expression. Hypoxic WJ-MSCs expressed higher mRNA levels of hypoxia inducible factors, notch receptors and notch downstream gene HES1. Gene expression profile of WJ-MSCs exposed to hypoxia and normoxia was compared and we identified a differential gene expression pattern where several stem cells markers and early mesodermal/endothelial genes such as DESMIN, CD34, ACTC were upregulated under hypoxia, suggesting that in vitro culturing of WJ-MSCs under hypoxic conditions leads to adoption of a mesodermal/endothelial fate. Thus, we demonstrate for the first time the effect of hypoxia on gene expression and growth kinetics of WJ-MSCs. Finally, although WJ-MSCs do not induce teratomas, under stressful and long-term culture conditions, MSCs can occasionally undergo transformation. Though there were no chromosomal abnormalities, certain transformation markers were upregulated in a few of the samples of WJ-MSCs under hypoxia.
PMCID: PMC2945278  PMID: 20877435
Hypoxia; Wharton's jelly; Mesenchymal stem cells (MSCs); Transcription; Transformation markers; Cell proliferation.
4.  SDF-1α/CXCR4 Axis Mediates The Migration of Mesenchymal Stem Cells to The Hypoxic-Ischemic Brain Lesion in A Rat Model 
Cell Journal (Yakhteh)  2015;16(4):440-447.
Objective
Transplantation of mesenchymal stem cells (MSCs) can promote functional recovery of the brain after hypoxic-ischemic brain damage (HIBD). However, the mechanism regulating MSC migration to a hypoxic-ischemic lesion is poorly understood. Interaction between stromal cell-derived factor-1α (SDF-1α) and its cognate receptor CXC chemokine receptor 4 (CXCR4) is crucial for homing and migration of multiple stem cell types. In this study, we investigate the potential role of SDF-1α/CXCR4 axis in mediating MSC migration in an HIBD model.
Materials and Methods
In this experimental study, we first established the animal model of HIBD using the neonatal rat. Bone marrow MSCs were cultured and labeled with 5-bromo-21-deoxyuridine (BrdU) after which 6×106 cells were intravenously injected into the rat. BrdU positive MSCs in the hippocampus were detected by immunohistochemical analyses. The expression of hypoxia-inducible factor-1α (HIF-1α) and SDF-1α in the hippocampus of hypoxic-ischemic rats was detected by Western blotting. To investigate the role of hypoxia and SDF-1α on migration of MSCs in vitro, MSCs isolated from normal rats were cultured in a hypoxic environment (PO2=1%). Migration of MSCs was detected by the transwell assay. The expression of CXCR4 was tested using Western blotting and flow cytometry.
Results
BrdU-labeled MSCs were found in the rat brain, which suggested that transplanted MSCs migrated to the site of the hypoxic-ischemic brain tissue. HIF-1α and SDF-1α significantly increased in the hippocampal formations of HIBD rats in a time-dependent manner. They peaked on day 7 and were stably expressed until day 21. Migration of MSCs in vitro was promoted by SDF-1α under hypoxia and inhibited by the CXCR4 inhibitor AMD3100. The expression of CXCR4 on MSCs was elevated by hypoxia stimulation as well as microdosage treatment of SDF-1α.
Conclusion
This observation illustrates that SDF-1α/CXCR4 axis mediate the migration of MSCs to a hypoxic-ischemic brain lesion in a rat model.
PMCID: PMC4297482
Mesenchymal Stem Cells; Migration; SDF-1α; CXCR 4
5.  Short-Term Exposure of Multipotent Stromal Cells to Low Oxygen Increases Their Expression of CX3CR1 and CXCR4 and Their Engraftment In Vivo 
PLoS ONE  2007;2(5):e416.
The ability of stem/progenitor cells to migrate and engraft into host tissues is key to their potential use in gene and cell therapy. Among the cells of interest are the adherent cells from bone marrow, referred to as mesenchymal stem cells or multipotent stromal cells (MSC). Since the bone marrow environment is hypoxic, with oxygen tensions ranging from 1% to 7%, we decided to test whether hypoxia can upregulate chemokine receptors and enhance the ability of human MSCs to engraft in vivo. Short-term exposure of MSCs to 1% oxygen increased expression of the chemokine receptors CX3CR1and CXCR4, both as mRNA and as protein. After 1-day exposure to low oxygen, MSCs increased in vitro migration in response to the fractalkine and SDF-1α in a dose dependent manner. Blocking antibodies for the chemokine receptors significantly decreased the migration. Xenotypic grafting into early chick embryos demonstrated cells from hypoxic cultures engrafted more efficiently than cells from normoxic cultures and generated a variety of cell types in host tissues. The results suggest that short-term culture of MSCs under hypoxic conditions may provide a general method of enhancing their engraftment in vivo into a variety of tissues.
doi:10.1371/journal.pone.0000416
PMCID: PMC1855077  PMID: 17476338
6.  Effect of acute hypoxia on CXCR4 gene expression in C57BL/6 mouse bone marrow-derived mesenchymal stem cells 
Background:
One of the most important stimuli in stem cell biology is oxygen. Chemokine receptor 4 (CXCR4) plays a crucial role in the migration and homing of stem cells. In this study, mesenchymal stem cells (MSCs) were exposed to 1% oxygen to investigate the effect of acute hypoxia on CXCR4 gene expression.
Materials and Methods:
MSCs were isolated from C57BL/6 mouse bone marrow and were identified and expanded in normoxic culture. Cells were incubated at 37°C under 1% hypoxic conditions for periods of 4, 8, 16, 24, and 48 h. After hypoxia preconditioning, the cells were placed in normoxic condition for 8 h to achieve cellular hypoxia-reoxygenation. To assess the level of CXCR4 gene expression, real-time quantitative reverse transcription-polymerase chain reaction was carried out for each group.
Results:
Data from statistical analysis illustrated that exposure of MSCs to acute hypoxic condition down-regulates CXCR4 expression with the maximum under-expression observed in 4 h (0.91 ± 0.107) and 8 h (50 ± 2.98) groups. Moreover, the relative gene expression of CXCR4 was decreased after hypoxia-reoxygenation by more than 80% in 4 h (0.136 ± 0.018) and 24 h (12.77 ± 0.707) groups.
Conclusion:
The results suggest that CXCR4 expression in MSCs decreases upon acute hypoxic stress. Furthermore, hypoxia-reoxygenated MSCs showed decreased expression of CXCR4, compared to cells subjected to acute hypoxia. This difference could have resulted from the cells being compatible with low oxygen metabolism. In summary, before the therapeutic application of MSCs, it should be regarded as a necessity to optimize the oxygen concentration in these cells, as it is a critical factor in modulating CXCR4 expression.
doi:10.4103/2277-9175.145682
PMCID: PMC4260281  PMID: 25538908
CXCR4; gene expression; hypoxia; mesenchymal stem cells
7.  Differential roles of hypoxia inducible factor subunits in multipotential stromal cells under hypoxic condition 
Journal of cellular biochemistry  2011;112(3):804-817.
Cell therapy with bone marrow multipotential stromal cells (MSCs) represents a promising approach to promote wound healing and tissue regeneration. MSCs expanded in vitro lose early progenitors with differentiation and therapeutic potentials under normoxic condition, whereas hypoxic condition promotes MSC self-renewal through preserving colony forming early progenitors and maintaining undifferentiated phenotypes. Hypoxia inducible factor (HIF) pathway is a crucial signaling pathway activated in hypoxic condition. We evaluated the roles of HIFs in MSC differentiation, colony formation, and paracrine activity under hypoxic condition. Hypoxic condition reversibly decreased osteogenic and adipogenic differentiation. Decrease of osteogenic differentiation depended on HIF pathway; whereas decrease of adipogenic differentiation depended on the activation of unfolded protein response (UPR), but not HIFs. Hypoxia-mediated increase of MSC colony formation was not HIF-dependent also. Hypoxic exposure increased secretion of VEGF, HGF and basic FGF in a HIF-dependent manner. These findings suggest that HIF has a limited, but pivotal role in enhancing MSC self-renewal and growth factor secretions under hypoxic condition.
doi:10.1002/jcb.22961
PMCID: PMC4110974  PMID: 21328454
Mesenchymal stem cells; Hypoxia-inducible factor; Hypoxia; Self renewal; Differentiation; oxygen
8.  Hypoxia Impedes Vasculogenesis of In Vitro Engineered Bone 
Tissue Engineering. Part A  2011;18(1-2):208-218.
To ensure the survival of engineered bone after implantation, we combined human endothelial colony forming cells (ECFCs) and multipotent stromal cells (MSCs) as a proof of concept in a co-culture model to create in vitro prevascularized bone constructs. We hypothesized that a hypoxic stimulus will contribute to prevascularization of engineered bone. Bone marrow-derived MSCs and ECFCs from human adult peripheral blood were allowed to form co-culture pellets containing ECFCs and MSCs (1:4) or MSCs only in controls. After culture under normoxia or hypoxia (5%), pellets were harvested and processed for immunohistochemistry of CD31, α-smooth muscle actin, and osteocalcin. Expression of vascular endothelial growth factor and SDF-1α was analyzed by PCR to elucidate their involvement in hypoxic stimulation of prevascularization. The normoxic condition in co-cultures of MSCs and ECFCs supported the formation and maintenance of prevascular structures, including organized CD31-positive cells embraced by differentiated mural cells. These structures failed to form in hypoxic conditions, thereby rejecting the hypothesis that hypoxia stimulates prevasculogenesis in three-dimensional engineered bone constructs. Further, the formation of prevascular structures was paralleled by increased SDF-1α expression. It is suggested that actual oxygen levels were below 5% in the hypoxic co-cultures, which prevented prevascular structure formation. In conclusion, our normoxic co-culture model containing cells from clinically relevant sources sustained simultaneous endothelial, smooth muscle, and osteogenic differentiation.
doi:10.1089/ten.tea.2010.0731
PMCID: PMC3246420  PMID: 21859278
9.  Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells 
BMC Cell Biology  2010;11:11.
Background
Human multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity.
Results
After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation.
Conclusion
Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.
doi:10.1186/1471-2121-11-11
PMCID: PMC2827377  PMID: 20109207
10.  Conditioned Medium from Hypoxic Bone Marrow-Derived Mesenchymal Stem Cells Enhances Wound Healing in Mice 
PLoS ONE  2014;9(4):e96161.
Growing evidence indicates that bone marrow-derived mesenchymal stem cells (BM-MSCs) enhance wound repair via paracrine. Because the extent of environmental oxygenation affects the innate characteristics of BM-MSCs, including their stemness and migration capacity, the current study set out to elucidate and compare the impact of normoxic and hypoxic cell-culture conditions on the expression and secretion of BM-MSC-derived paracrine molecules (e.g., cytokines, growth factors and chemokines) that hypothetically contribute to cutaneous wound healing in vivo. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses of normoxic and hypoxic BM-MSCs and their conditioned medium fractions showed that the stem cells expressed and secreted significantly higher amounts of basic fibroblast growth factor (bFGF),vascular endothelial growth factor A (VEGF-A) interleukin 6 (IL-6) and interleukin 8 (IL-8) under hypoxic conditions. Moreover, hypoxic BM-MSC-derived conditioned medium (hypoCM) vs. normoxic BM-MSC-derived conditioned medium (norCM) or vehicle control medium significantly enhanced the proliferation of keratinocytes, fibroblasts and endothelial cells, the migration of keratinocytes, fibroblasts, endothelial cells and monocytes, and the formation of tubular structures by endothelial cells cultured on Matrigel matrix. Consistent with these in vitro results, skin wound contraction was significantly accelerated in Balb/c nude mice treated with topical hypoCM relative to norCM or the vehicle control. Notably increased in vivo cell proliferation, neovascularization as well as recruitment of inflammatory macrophages and evidently decreased collagen I, and collagen III were also found in the hypoCM-treated group. These findings suggest that BM-MSCs promote murine skin wound healing via hypoxia-enhanced paracrine.
doi:10.1371/journal.pone.0096161
PMCID: PMC4004560  PMID: 24781370
11.  Priming with Ligands Secreted by Human Stromal Progenitor Cells Promotes Grafts of Cardiac Stem/Progenitor Cells after MyocarDial Infarction 
Stem cells (Dayton, Ohio)  2014;32(3):674-683.
Transplantation of culture-expanded adult stem/progenitor cells often results in poor cellular engraftment, survival, and migration into sites of tissue injury. Mesenchymal cells including fibroblasts and stromal cells secrete factors that protect injured tissues, promote tissue repair, and support many types of stem/progenitor cells in culture. We hypothesized that secreted factors in conditioned medium (CdM) from adult bone marrow-derived multipotent stromal cells (MSCs) could be used to prime adult cardiac stem/progenitor cells (CSCs/CPCs) and improve graft success after myocardial infarction (MI). Incubation of adult rat CPCs in CdM from human MSCs isolated by plastic adherence or by magnetic sorting against CD271 (a.k.a., p75 low-affinity nerve growth factor receptor; p75MSCs) induced phosphorylation of STAT3 and Akt in CPCs, supporting their proliferation under normoxic conditions and survival under hypoxic conditions (1% oxygen). Priming CSCs with 30x p75MSC CdM for 30 min prior to transplantation into sub-epicardial tissue 1 day after MI markedly increased engraftment compared with vehicle priming. Screening CdM with neutralizing/blocking antibodies identified Connective Tissue Growth Factor (CTGF) and Insulin as key factors in p75MSC CdM that protected CPCs. Human CTGF peptide (CTGF-D4) and Insulin synergistically promoted CPC survival during hypoxia in culture. Similar to CdM priming, priming of CSCs with CTGF-D4 and Insulin for 30 min prior to transplantation promoted robust engraftment, survival and migration of CSC derivatives at 1 week and 1 month after MI. Our results indicate that short-term priming of human CSCs with CTGF-D4 and Insulin may improve graft success and cardiac regeneration in patients with MI.
doi:10.1002/stem.1546
PMCID: PMC3966427  PMID: 24022988
Stem cells; progenitor cells; MSCs; CSCs/CPCs; stromal cells; paracrine; CTGF; Insulin
12.  Inositol pyrophosphates mediate the effects of aging on bone marrow mesenchymal stem cells by inhibiting Akt signaling 
Introduction
Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been proposed as an ideal autologous stem cell source for cell-based therapy for myocardial infarction (MI). However, decreased viability and impaired function of aged MSCs hampered the therapeutic efficacy of engrafted MSCs, and the underlying mechanisms remain unclarified. Here, we investigated the role of inositol phosphates 6 kinase (IP6Ks) inhibition on the therapeutic efficacy of BM-MSCs and its underlying mechanism.
Methods
BM-MSCs isolated from young (8-week-old) or aged (18-month-old) donor male C57BL/6 mice, were subjected to hypoxia and serum deprivation (H/SD) injury with or without administration of inositol phosphates 6 kinase (IP6Ks) inhibitor TNP (10 μM). MSC apoptosis induced by H/SD was determined by flow cytometry and TUNEL assays. Protein expressions were evaluated by Western blot assay. Furthermore, the paracrine effects of MSCs were measured by reverse transcriptase–polymerized chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses.
Results
Aged BM-MSCs exhibited more Inositol pyrophosphate 7 (IP7) production, compared with young BM-MSCs. Meanwhile, the expression of phospho-Akt (Thr308) was significantly decreased in the aged MSCs, resulting in enhanced Bad activation and decreased Bax/Bcl-2 ratio. Moreover, the apoptosis in aged BM-MSCs was increased, compared with young BM-MSCs. Furthermore, TNP administration significantly inhibited IP7 production and increased the phosphorylation of Akt under both normoxic and hypoxic conditions. Meanwhile, IP6Ks inhibition reduced apoptotic index of aged MSCs, associated with decreased expressions of pro-apoptotic proteins Bax and Bad and increased anti-apoptotic protein Bcl-2. The expressions of angiogenic factors, including VEGF, bFGF, IGF-1 and HGF, were decreased in MSCs from aged mice. In addition, TNP administration enhanced the paracrine efficiency of aged BM-MSCs under normoxic and hypoxic conditions.
Conclusions
This study demonstrates for the first time that IP6Ks and IP7 play critical role in the aging related vulnerability to hypoxic injury and impaired paracrine efficiency of BM-MSCs, which is associated with impaired Akt activation.
doi:10.1186/scrt431
PMCID: PMC4055148  PMID: 24670364
13.  shRNA-Mediated Decreases in c-Met Levels Affect the Differentiation Potential of Human Mesenchymal Stem Cells and Reduce Their Capacity for Tissue Repair 
Tissue Engineering. Part A  2010;16(8):2627-2639.
Mesenchymal stem cells/marrow stromal cells (MSC) are adult multipotent cells that can augment tissue repair. We previously demonstrated that culturing MSC in hypoxic conditions causes upregulation of the hepatocyte growth factor (HGF) receptor c-Met, allowing them to respond more robustly to HGF. MSC preconditioned in hypoxic environments contributed to restoration of blood flow after an ischemic injury more rapidly than MSC cultured in normoxic conditions. We now investigated the specific role of HGF/c-Met signaling in MSC function. An shRNA-mediated knockdown (KD) of c-Met in MSC did not alter their phenotypic profile, proliferation, or viability in vitro. However, we determined that while HGF/c-Met signaling does not play a role in the adipogenic differentiation of the cells, the disruption of this signaling pathway inhibited the ability of MSC to differentiate into the osteogenic and chondrogenic lineages. We next assessed the impact of c-Met KD on human MSC function in a xenogeneic hindlimb ischemia injury model. A 70% KD of c-Met in MSC resulted in a significant decrease in their capacity to regenerate blood flow to the ischemic limb, as compared to the MSC transduced with control shRNA. MSC with only a 60% KD of c-Met exhibited an intermediate capacity to restore blood flow, suggesting that MSC function is sensitive to the dosage of c-Met signaling. The current study highlights the significance of HGF/c-Met signaling in the capacity of MSC to restore blood flow after an ischemic injury and in their ability to differentiate into the osteogenic and chondrogenic lineages.
doi:10.1089/ten.tea.2009.0363
PMCID: PMC2947453  PMID: 20367286
14.  Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent status 
BMC Cell Biology  2011;12:12.
Background
In the bone marrow, hematopietic and mesenchymal stem cells form a unique niche in which the oxygen tension is low. Hypoxia may have a role in maintaining stem cell fate, self renewal and multipotency. However, whereas most studies addressed the effect of transient in vitro exposure of MSC to hypoxia, permanent culture under hypoxia should reflect the better physiological conditions.
Results
Morphologic studies, differentiation and transcriptional profiling experiments were performed on MSC cultured in normoxia (21% O2) versus hypoxia (5% O2) for up to passage 2. Cells at passage 0 and at passage 2 were compared, and those at passage 0 in hypoxia generated fewer and smaller colonies than in normoxia. In parallel, MSC displayed (>4 fold) inhibition of genes involved in DNA metabolism, cell cycle progression and chromosome cohesion whereas transcripts involved in adhesion and metabolism (CD93, ESAM, VWF, PLVAP, ANGPT2, LEP, TCF1) were stimulated. Compared to normoxic cells, hypoxic cells were morphologically undifferentiated and contained less mitochondrias. After this lag phase, cells at passage 2 in hypoxia outgrew the cells cultured in normoxia and displayed an enhanced expression of genes (4-60 fold) involved in extracellular matrix assembly (SMOC2), neural and muscle development (NOG, GPR56, SNTG2, LAMA) and epithelial development (DMKN). This group described herein for the first time was assigned by the Gene Ontology program to "plasticity".
Conclusion
The duration of hypoxemia is a critical parameter in the differentiation capacity of MSC. Even in growth promoting conditions, hypoxia enhanced a genetic program that maintained the cells undifferentiated and multipotent. This condition may better reflect the in vivo gene signature of MSC, with potential implications in regenerative medicine.
doi:10.1186/1471-2121-12-12
PMCID: PMC3073900  PMID: 21450070
15.  SDF-1α Secreted by Human CD133-Derived Multipotent Stromal Cells Promotes Neural Progenitor Cell Survival Through CXCR7 
Stem Cells and Development  2010;20(6):1021-1029.
We recently reported that concentrated conditioned medium (CdM) from human CD133-derived bone marrow progenitor cells (CD133 CdM) was neuroprotective after stroke. Here we identify stromal-derived factor 1 alpha (SDF-1) as a potential neuroprotective candidate in CD133 CdM by interrogating the transcriptional responses of CD133-derived multipotent stromal cells (CD133dMSCs) after cell injection into the ischemic brain. Human SDF-1 mRNA was upregulated 79-fold by CD133dMSCs when injected into the stroke peri-infarct area compared with cells injected into the uninjured parenchyma of sham-operated animals. In cell protection assays, we replaced the typical growth medium in mouse neural progenitor cell (mNPC) cultures with serum-free CD133 CdM immediately before exposure to hypoxia (1% oxygen) for 48 h. CD133 CdM significantly increased the survival of mNPCs during hypoxia exposure and growth factor withdrawal. To determine whether MSC-secreted SDF-1 influenced mNPC survival, we used lentiviral short hairpin RNA against SDF1 (shSDF-1) to knockdown SDF-1 expression in CD133dMSCs. The CdM generated from shSDF-1-treated cells had a 94% decrease in secreted SDF-1 and was significantly less protective for mNPCs when compared with control CdM from CD133dMSCs transduced with scrambled short hairpin RNA. Pharmacological inhibition of the 2 known SDF-1 receptors, CXCR4 and CXCR7, revealed that only CXCR7 activity was functionally linked to survival signaling in mNPCs during hypoxia exposure. Treatment of mNPCs with CD133 CdM and CXCR7 inhibitor decreased mNPC viability by 36.5% ± 12.8% and decreased cell number by 21% ± 6.7% compared with dimethyl sulfoxide treated controls. These data indicate that SDF-1 is a key neuroprotective cytokine secreted by CD133dMSCs that protects mNPCs through CXCR7.
doi:10.1089/scd.2010.0198
PMCID: PMC3102777  PMID: 20854168
16.  Lung-Derived Mesenchymal Stromal Cell Post-Transplantation Survival, Persistence, Paracrine Expression, and Repair of Elastase-Injured Lung 
Stem Cells and Development  2011;20(10):1779-1792.
While multipotent mesenchymal stromal cells have been recently isolated from adult lung (L-MSCs), there is very limited data on their biological properties and therapeutic potential in vivo. How L-MSCs compare with bone marrow-derived MSCs (BM-MSCs) is also unclear. In this study, we characterized L-MSC phenotype, clonogenicity, and differentiation potential, and compared L-MSCs to BM-MSCs in vivo survival, retention, paracrine gene expression, and repair or elastase injury after transplantation. L-MSCs were highly clonogenic, frequently expressed aldehyde dehydrogenase activity, and differentiated into osteocytes, chondrocytes, adipocytes, myofibroblasts, and smooth muscle cells. After intravenous injection (2 h), L-MSCs showed greater survival than BM-MSCs; similarly, L-MSCs were significantly more resistant than BM-MSCs to anchorage independent culture (4 h) in vitro. Long after transplantation (4 or 32 days), a significantly higher number of CD45neg L-MSCs were retained than BM-MSCs. By flow cytometry, L-MSCs expressed more intercellular adhesion molecule-1 (ICAM-1), platelet derived growth factor receptor alpha (PDGFRα), and integrin α2 than BM-MSCs; these proteins were found to modulate endothelial adherence, directional migration, and migration across Matrigel in L-MSCs. Further, L-MSCs with low ICAM-1 showed poorer lung retention and higher phagocytosis in vivo. Compared with BM-MSCs, L-MSCs expressed higher levels of several transcripts (e.g., Ccl2, Cxcl2, Cxcl10, IL-6, IL-11, Hgf, and Igf2) in vitro, although gene expression in vivo was increased by L-MSCs and BM-MSCs equivalently. Accordingly, both L-MSCs and BM-MSCs reduced elastase injury to the same extent. This study demonstrates that tissue-specific L-MSCs possess mechanisms that enhance their lung retention after intravenous transplantation, and produce substantial healing of elastase injury comparable to BM-MSCs.
doi:10.1089/scd.2011.0105
PMCID: PMC3182034  PMID: 21585237
17.  The Primary Cilium as a Biomarker in the Hypoxic Adaptation of Bone Marrow-Derived Mesenchymal Stromal Cells: A Role for the Secreted Frizzled-Related Proteins 
Biomarker Insights  2011;6:107-118.
A pivotal role in guiding mesenchymal stem cell (MSC) differentiation has recently been attributed to the primary cilium. This solitary, non-motile microtubule-based organelle emerging from the cell surface acts as a sensorial membrane structure reflecting developmental and adaptive processes associated with pathologies including human cystic kidney disease, skeletal malformations, obesity and cancer. Given that the intrinsic hypoxic adaptation of MSC remains poorly understood within ischemic tissues or hypoxic tumours, we questioned whether the hypoxia inducible factor-1α (HIF-1α) might be a downstream effector regulating cilium maintenance. We show that murine bone marrow-derived MSC cultured under hypoxic conditions (1.2% O2) lose their primary cilia in a time-dependent manner. Gene silencing of HIF-1α prevented cilia loss in hypoxic cultures, and generation of MSC expressing a constitutively active HIF-1α (MSC-HIF) was found to decrease primary cilium formation. A Wnt pathway-related gene expression array was also performed on MSC-HIF and indicated that the secreted Frizzled-related proteins (sFRP)-1, -3 and -4 were down-regulated, while sFRP-2 was up-regulated. Overexpression of recombinant sFRP-2 or gene silencing of sFRP-1, -3 and -4 in MSC led to primary cilium disruption. These results indicate a molecular signalling mechanism for the hypoxic disruption of the primary cilium in MSC involving an HIF-1α/sFRP axis. This mechanism contributes to our understanding of the adaptive processes possibly involved in the oncogenic transformation and tumour-supporting potential of MSC. Our current observations also open up the possibility for the primary cilia to serve as a biomarker in MSC adaptation to low oxygen tension within (patho)physiological microenvironments.
doi:10.4137/BMI.S8247
PMCID: PMC3201088  PMID: 22084569
cilia; hypoxia; mesenchymal stem cells; hypoxia-inducible factor (HIF)
18.  Hypoxia mediated isolation and expansion enhances the chondrogenic capacity of bone marrow mesenchymal stromal cells 
Introduction
The capacity of bone marrow mesenchymal stromal cells (BMSCs) to be induced into chondrocytes has drawn much attention for cell-based cartilage repair. BMSCs represent a small proportion of cells of the bone marrow stromal compartment and, thus, culture expansion is a necessity for therapeutic use. However, there is no consensus on how BMSCs should be isolated nor expanded to maximize their chondrogenic potential. During embryonic development pluripotent stem cells differentiate into chondrocytes and form cartilage in a hypoxic microenvironment.
Methods
Freshly harvested human BMSCs were isolated and expanded from the aspirates of six donors, under either hypoxic conditions (3% O2) or normoxic conditions (21% O2). A colony-forming unit fibroblastic (Cfu-f) assay was used to determine the number of cell colonies developed from each donor. BMSCs at passage 2 (P2) were characterized by flow cytometry for the phenotypic expression of cell surface markers on mesenchymal stem cells. BMSCs at P2 were subsequently cultured in vitro as three-dimensional cell pellets in a defined serum-free chondrogenic medium under normoxic and hypoxic conditions. Chondrogenic differentiation of the BMSCs was characterized by biochemical and histological methods and by quantitative gene-expression analysis.
Results
After 14 days of culture, the number of BMSC colonies developed under hypoxia was generally higher (8% to 38% depending on donor) than under normoxia. BMSCs were positive for the cell surface markers CD13, CD29, CD44, CD73, CD90, CD105 and CD151, and negative for CD34. Regardless of the oxygen tension during pellet culture, hypoxia-expanded BMSC pellets underwent a more robust chondrogenesis than normoxia-expanded BMSC pellets after three weeks of culture, as judged by increased glycosaminoglycan synthesis and Safranin O staining, along with increased mRNA expression of aggrecan, collagen II and Sox9. Hypoxic conditions enhanced the mRNA expression of hypoxia inducible factor-2 alpha (HIF-2α) but suppressed the mRNA expression of collagen X in BMSC pellet cultures regardless of the oxygen tension during BMSC isolation and propagation.
Conclusions
Taken together, our data demonstrate that isolation and expansion of BMSCs under hypoxic conditions augments the chondrogenic potential of BMSCs. This suggests that hypoxia-mediated isolation and expansion of BMSCs may improve clinical applications of BMSCs for cartilage repair.
doi:10.1186/scrt100
PMCID: PMC3392769  PMID: 22385573
Chondrogenesis; chondrocytes; hypoxia; bone marrow stem cells; tissue engineering; cartilage repair
19.  Hypoxic Conditioned Medium from Human Amniotic Fluid-Derived Mesenchymal Stem Cells Accelerates Skin Wound Healing through TGF-β/SMAD2 and PI3K/Akt Pathways 
In a previous study, we isolated human amniotic fluid (AF)-derived mesenchymal stem cells (AF-MSCs) and utilized normoxic conditioned medium (AF-MSC-norCM) which has been shown to accelerate cutaneous wound healing. Because hypoxia enhances the wound healing function of mesenchymal stem cell-conditioned medium (MSC-CM), it is interesting to explore the mechanism responsible for the enhancement of wound healing function. In this work, hypoxia not only increased the proliferation of AF-MSCs but also maintained their constitutive characteristics (surface marker expression and differentiation potentials). Notably, more paracrine factors, VEGF and TGF-β1, were secreted into hypoxic conditioned medium from AF-MSCs (AF-MSC-hypoCM) compared to AF-MSC-norCM. Moreover, AF-MSC-hypoCM enhanced the proliferation and migration of human dermal fibroblasts in vitro, and wound closure in a skin injury model, as compared to AF-MSC-norCM. However, the enhancement of migration of fibroblasts accelerated by AF-MSC-hypoCM was inhibited by SB505124 and LY294002, inhibitors of TGF-β/SMAD2 and PI3K/AKT, suggesting that AF-MSC-hypoCM-enhanced wound healing is mediated by the activation of TGF-β/SMAD2 and PI3K/AKT. Therefore, AF-MSC-hypoCM enhances wound healing through the increase of hypoxia-induced paracrine factors via activation of TGF-β/SMAD2 and PI3K/AKT pathways.
doi:10.3390/ijms15010605
PMCID: PMC3907828  PMID: 24398984
hypoxia; amniotic fluid-derived mesenchymal stem cells (AF-MSCs); wound healing; PI3K/AKT; TGF-β/SMAD2
20.  Adipose Tissue-Derived Multipotent Stromal Cells Have a Higher Immunomodulatory Capacity Than Their Bone Marrow-Derived Counterparts 
This study systematically compared the immunomodulatory capacities of adipose tissue-derived multipotent stromal cells (AT-MSCs) and bone marrow-derived multipotent stromal cells (BM-MSCs) derived from age-matched donors. It was found that BM-MSCs and AT-MSCs show functionally similar immunomodulatory effects, but with a different dose-response curve, in favor of AT-MSCs. AT-MSCs can be considered as a good alternative to BM-MSCs for immunomodulatory therapy.
Adipose tissue-derived multipotent stromal cells (AT-MSCs) are studied as an alternative to bone marrow-derived multipotent stromal cells (BM-MSCs) for immunomodulatory treatment. In this study, we systematically compared the immunomodulatory capacities of BM-MSCs and AT-MSCs derived from age-matched donors. We found that BM-MSCs and AT-MSCs share a similar immunophenotype and capacity for in vitro multilineage differentiation. BM-MSCs and AT-MSCs showed comparable immunomodulatory effects as they were both able to suppress proliferation of stimulated peripheral blood mononuclear cells and to inhibit differentiation of monocyte-derived immature dendritic cells. However, at equal cell numbers, the AT-MSCs showed more potent immunomodulatory effects in both assays as compared with BM-MSCs. Moreover, AT-MSCs showed a higher level of secretion of cytokines that have been implicated in the immunomodulatory modes of action of multipotent stromal cells, such as interleukin-6 and transforming growth factor-β1. This is correlated with higher metabolic activity of AT-MSCs compared with BM-MSCs. We conclude that the immunomodulatory capacities of BM-MSCs and AT-MSCs are similar, but that differences in cytokine secretion cause AT-MSCs to have more potent immunomodulatory effects than BM-MSCs. Therefore, lower numbers of AT-MSCs evoke the same level of immunomodulation. These data indicate that AT-MSCs can be considered as a good alternative to BM-MSCs for immunomodulatory therapy.
doi:10.5966/sctm.2012-0184
PMCID: PMC3673757  PMID: 23694810
Adult human bone marrow; Cellular therapy; Bone marrow; Bone marrow stromal cells; Immunosuppression; Marrow stromal stem cells; Mesenchymal stem cells
21.  Nestin expression in mesenchymal stromal cells: regulation by hypoxia and osteogenesis 
BMC Veterinary Research  2014;10:173.
Background
The intermediate filament protein nestin is used as a marker for neural stem cells, and its expression is inversely correlated with cellular differentiation. More recently, nestin expression has also been described in other cell types including multipotential mesenchymal stromal cells (MSCs). In this study, we examined the expression of nestin in equine, canine and human bone marrow-derived MSCs undergoing osteogenic differentiation, to determine whether nestin levels were attenuated as the cells acquired a more mature phenotype. In addition, the expression of nestin may be under the influence of cellular hypoxia, as nestin expression is known to increase in areas of ischemic tissue damage. Therefore, we also examined the effects of hypoxia on expression of nestin in human MSCs and examined a role for hypoxia inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in the response. Additionally, we quantified the temporal expression of nestin in the fracture callus during bone regeneration, a site that has been characterized as hypoxic.
Results
There were no significant changes in nestin expression in MSCs during osteogenic differentiation. There was a significant increase in expression of nestin mRNA and protein in human MSCs in response to hypoxia (1% O2) or the chemical hypoxia mimetic desferroxamine. This may be due to upregulation of VEGF under hypoxia, as treatment of cells with the VEGF receptor antagonist CPO-P11 attenuated hypoxia-induced nestin expression. A significant increase in nestin mRNA expression was observed in the fracture callus of mice three and seven days post fracture.
Conclusions
Nestin was not a selective marker for MSCs, as its expression was maintained during osteogenic differentiation, in all species examined. Furthermore our data suggest that nestin expression can be induced by hypoxia, and that this increase in nestin is partially regulated by HIF-1α and VEGF. Interestingly, nestin levels were significantly upregulated at the fracture site. Further studies are required to understand the role of nestin in bone cell biology and ultimately bone regeneration.
doi:10.1186/s12917-014-0173-z
PMCID: PMC4236815  PMID: 25088159
Nestin; Mesenchymal stromal cells; Canine; Equine; Human; Fracture callus; Osteogenesis; VEGF
22.  Human Bone Marrow-Derived Mesenchymal Stem Cells Display Enhanced Clonogenicity but Impaired Differentiation With Hypoxic Preconditioning 
The use of hypoxia was investigated as a preconditioning agent and in differentiating cultures to enhance mesenchymal stem cell (MSC) function. Findings suggest that hypoxic preconditioning may blunt the differentiation potential of MSCs, compromising their utility for regenerative tissue engineering. Exposure to hypoxia during differentiation (post-normoxic expansion), however, appears to result in a greater quantity of functional osteoblasts and chondrocytes and ultimately a larger quantity of high-quality differentiated tissue.
Stem cells are promising candidate cells for regenerative applications because they possess high proliferative capacity and the potential to differentiate into other cell types. Mesenchymal stem cells (MSCs) are easily sourced but do not retain their proliferative and multilineage differentiative capabilities after prolonged ex vivo propagation. We investigated the use of hypoxia as a preconditioning agent and in differentiating cultures to enhance MSC function. Culture in 5% ambient O2 consistently enhanced clonogenic potential of primary MSCs from all donors tested. We determined that enhanced clonogenicity was attributable to increased proliferation, increased vascular endothelial growth factor secretion, and increased matrix turnover. Hypoxia did not impact the incidence of cell death. Application of hypoxia to osteogenic cultures resulted in enhanced total mineral deposition, although this effect was detected only in MSCs preconditioned in normoxic conditions. Osteogenesis-associated genes were upregulated in hypoxia, and alkaline phosphatase activity was enhanced. Adipogenic differentiation was inhibited by exposure to hypoxia during differentiation. Chondrogenesis in three-dimensional pellet cultures was inhibited by preconditioning with hypoxia. However, in cultures expanded under normoxia, hypoxia applied during subsequent pellet culture enhanced chondrogenesis. Whereas hypoxic preconditioning appears to be an excellent way to expand a highly clonogenic progenitor pool, our findings suggest that it may blunt the differentiation potential of MSCs, compromising their utility for regenerative tissue engineering. Exposure to hypoxia during differentiation (post-normoxic expansion), however, appears to result in a greater quantity of functional osteoblasts and chondrocytes and ultimately a larger quantity of high-quality differentiated tissue.
doi:10.5966/sctm.2013-0079
PMCID: PMC3925051  PMID: 24436440
Mesenchymal stem cells; Hypoxia; Colony-forming units assay; Chondrogenesis; Osteogenesis; Adipogenesis
23.  Constitutive stabilization of hypoxia-inducible factor alpha selectively promotes the self-renewal of mesenchymal progenitors and maintains mesenchymal stromal cells in an undifferentiated state 
With the increasing use of culture-expanded mesenchymal stromal cells (MSCs) for cell therapies, factors that regulate the cellular characteristics of MSCs have been of major interest. Oxygen concentration has been shown to influence the functions of MSCs, as well as other normal and malignant stem cells. However, the underlying mechanisms of hypoxic responses and the precise role of hypoxia-inducible factor-1α (Hif-1α), the master regulatory protein of hypoxia, in MSCs remain unclear, due to the limited span of Hif-1α stabilization and the complex network of hypoxic responses. In this study, to further define the significance of Hif-1α in MSC function during their self-renewal and terminal differentiation, we established adult bone marrow (BM)-derived MSCs that are able to sustain high level expression of ubiquitin-resistant Hif-1α during such long-term biological processes. Using this model, we show that the stabilization of Hif-1α proteins exerts a selective influence on colony-forming mesenchymal progenitors promoting their self-renewal and proliferation, without affecting the proliferation of the MSC mass population. Moreover, Hif-1α stabilization in MSCs led to the induction of pluripotent genes (oct-4 and klf-4) and the inhibition of their terminal differentiation into osteogenic and adipogenic lineages. These results provide insights into the previously unrecognized roles of Hif-1α proteins in maintaining the primitive state of primary MSCs and on the cellular heterogeneities in hypoxic responses among MSC populations.
doi:10.1038/emm.2013.87
PMCID: PMC3789268  PMID: 24071737
Hif-1α; hypoxia; MSC; self-renewal
24.  SIRT1 Inhibition Affects Angiogenic Properties of Human MSCs 
BioMed Research International  2014;2014:783459.
Human mesenchymal stem cells (hMSCs) are attractive for clinical and experimental purposes due to their capability of self-renewal and of differentiating into several cell types. Autologous hMSCs transplantation has been proven to induce therapeutic angiogenesis in ischemic disorders. However, the molecular mechanisms underlying these effects remain unclear. A recent report has connected MSCs multipotency to sirtuin families, showing that SIRT1 can regulate MSCs function. Furthermore, SIRT1 is a critical modulator of endothelial angiogenic functions. Here, we described the generation of an immortalized human mesenchymal bone marrow-derived cell line and we investigated the angiogenic phenotype of our cellular model by inhibiting SIRT1 by both the genetic and pharmacological level. We first assessed the expression of SIRT1 in hMSCs under basal and hypoxic conditions at both RNA and protein level. Inhibition of SIRT1 by sirtinol, a cell-permeable inhibitor, or by specific sh-RNA resulted in an increase of premature-senescence phenotype, a reduction of proliferation rate with increased apoptosis. Furthermore, we observed a consistent reduction of tubule-like formation and migration and we found that SIRT1 inhibition reduced the hypoxia induced accumulation of HIF-1α protein and its transcriptional activity in hMSCs. Our findings identify SIRT1 as regulator of hypoxia-induced response in hMSCs and may contribute to the development of new therapeutic strategies to improve regenerative properties of mesenchymal stem cells in ischemic disorders through SIRT1 modulation.
doi:10.1155/2014/783459
PMCID: PMC4163475  PMID: 25243179
25.  Hypoxia reduces CD138 expression and induces an immature and stem cell-like transcriptional program in myeloma cells 
International Journal of Oncology  2013;43(6):1809-1816.
Although CD138 expression is a hallmark of plasma cells and myeloma cells, reduced CD138 expression is occasionally found. However, the mechanisms underlying CD138 downregulation in myeloma cells remain unclear. Previous reports suggest that the bone marrow microenvironment may contribute to CD138 downregulation. Among various factors in the tumor microenvironment, hypoxia is associated with tumor progression, poor clinical outcomes, dedifferentiation and the formation of cancer stem cell niches in solid tumors. Since recent findings showed that progression of multiple myeloma (MM) delivers hypoxia within the bone marrow, we hypothesized that CD138 expression may be regulated by hypoxia. In the present study, we examined whether the expression of CD138 and transcription factors occurred in myeloma cells under hypoxic conditions. MM cell lines (KMS-12BM and RPMI 8226) were cultured under normoxic or hypoxic conditions for up to 30 days. Changes in the phenotype and the expression of surface antigens and transcription factors were analyzed using flow cytometry, RT-PCR and western blotting. All-trans retinoic acid (ATRA) was used to examine the phenotypic changes under hypoxic conditions. The expression levels of CD138, CS1 and plasma cell-specific transcription factors decreased under hypoxic conditions, while those of CD20, CXCR4 and B cell-specific transcription factors increased compared with those under normoxic conditions. Stem cell-specific transcription factors were upregulated under hypoxic conditions, while no difference was observed in ALDH activity. The reduced CD138 expression under hypoxic conditions recovered when cells were treated with ATRA, even under hypoxic conditions, along with decreases in the expression of stem cell-specific transcription factor. Interestingly, ATRA treatment sensitized MM cells to bortezomib under hypoxia. We propose that hypoxia induces immature and stem cell-like transcription phenotypes in myeloma cells. Taken together with our previous observation that decreased CD138 expression is correlated with disease progression, the present data suggest that a hypoxic microenvironment affects the phenotype of MM cells, which may correlate with disease progression.
doi:10.3892/ijo.2013.2134
PMCID: PMC3834117  PMID: 24126540
myeloma; hypoxia; CD138; ATRA

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