Hypoxia or in situ normoxia: The stem cell paradigm

Although O₂ concentrations are considerably lowered in vivo, depending on the tissue and cell population in question (some cells need almost anoxic environment for their maintenance) the cell and tissue cultures are usually performed at atmospheric O₂ concentration (20–21%). As an instructive example, the relationship between stem cells and micro‐environmental/culture oxygenation has been recapitulated. The basic principle of stem cell biology, “the generation‐age hypothesis,” and hypoxic metabolic properties of stem cells are considered in the context of the oxygen‐dependent evolution of life and its transposition to ontogenesis and development. A hypothesis relating the self‐renewal with the anaerobic and hypoxic metabolic properties of stem cells and the actual O₂ availability is elaborated (“oxygen stem cell paradigm”). Many examples demonstrated that the cellular response is substantially different at atmospheric O₂ concentration when compared to lower O₂ concentrations which better approximate the physiologic situation. These lower O₂ concentrations, traditionally called “hypoxia” represent, in fact, an in situ normoxia, and should be used in experimentation to get an insight of the real cell/cytokine physiology. The revision of our knowledge on cell/cytokine physiology, which has been acquired ex vivo at non physiological atmospheric (20–21%) O₂ concentrations representing a hyperoxic state for most primate cells, has thus become imperious. J. Cell. Physiol. 219: 271–275, 2009. © 2009 Wiley‐Liss, Inc.     

Quantitative phosphoproteomic analysis of prion-infected neuronal cells

Following cultivation of distinct mesenchymal stem cell (MSC) populations derived from human umbilical cord under hypoxic conditions (between 1.5% to 5% oxygen (O₂)) revealed a 2- to 3-fold reduced oxygen consumption rate as compared to the same cultures at normoxic oxygen levels (21% O₂). A simultaneous measurement of dissolved oxygen within the culture media from 4 different MSC donors ranged from 15 μmol/L at 1.5% O₂ to 196 μmol/L at normoxic 21% O₂. The proliferative capacity of the different hypoxic MSC populations was elevated as compared to the normoxic culture. This effect was paralleled by a significantly reduced cell damage or cell death under hypoxic conditions as evaluated by the cellular release of LDH whereby the measurement of caspase3/7 activity revealed little if any differences in apoptotic cell death between the various cultures. The MSC culture under hypoxic conditions was associated with the induction of hypoxia-inducing factor-alpha (HIF-1α) and an elevated expression of energy metabolism-associated genes including GLUT-1, LDH and PDK1. Concomitantly, a significantly enhanced glucose consumption and a corresponding lactate production could be observed in the hypoxic MSC cultures suggesting an altered metabolism of these human stem cells within the hypoxic environment.     

Cartilage Tissue Engineering Using Dermis Isolated Adult Stem Cells: The Use of Hypoxia during Expansion versus Chondrogenic Differentiation

Dermis isolated adult stem (DIAS) cells, a subpopulation of dermis cells capable of chondrogenic differentiation in the presence of cartilage extracellular matrix, are a promising source of autologous cells for tissue engineering. Hypoxia, through known mechanisms, has profound effects on in vitro chondrogenesis of mesenchymal stem cells and could be used to improve the expansion and differentiation processes for DIAS cells. The objective of this study was to build upon the mechanistic knowledge of hypoxia and translate it to tissue engineering applications to enhance chondrogenic differentiation of DIAS cells through exposure to hypoxic conditions (5% O₂) during expansion and/or differentiation. DIAS cells were isolated and expanded in hypoxic (5% O₂) or normoxic (20% O₂) conditions, then differentiated for 2 weeks in micromass culture on chondroitin sulfate-coated surfaces in both environments. Monolayer cells were examined for proliferation rate and colony forming efficiency. Micromasses were assessed for cellular, biochemical, and histological properties. Differentiation in hypoxic conditions following normoxic expansion increased per cell production of collagen type II 2.3 fold and glycosaminoglycans 1.2 fold relative to continuous normoxic culture (p<0.0001). Groups expanded in hypoxia produced 51% more collagen and 23% more GAGs than those expanded in normoxia (p<0.0001). Hypoxia also limited cell proliferation in monolayer and in 3D culture. Collectively, these data show hypoxic differentiation following normoxic expansion significantly enhances chondrogenic differentiation of DIAS cells, improving the potential utility of these cells for cartilage engineering.     

Moderate Exercise Mitigates the Detrimental Effects of Aging on Tendon Stem Cells

Aging is known to cause tendon degeneration whereas moderate exercise imparts beneficial effects on tendons. Since stem cells play a vital role in maintaining tissue integrity, in this study we aimed to define the effects of aging and moderate exercise on tendon stem/progenitor cells (TSCs) using in vitro and in vivo models. TSCs derived from aging mice (9 and 24 months) proliferated significantly slower than TSCs obtained from young mice (2.5 and 5 months). In addition, expression of the stem cell markers Oct-4, nucleostemin (NS), Sca-1 and SSEA-1 in TSCs decreased in an age-dependent manner. Interestingly, moderate mechanical stretching (4%) of aging TSCs in vitro significantly increased the expression of the stem cell marker, NS, but 8% stretching decreased NS expression. Similarly, 4% mechanical stretching increased the expression of Nanog, another stem cell marker, and the tenocyte-related genes, collagen I and tenomodulin. However, 8% stretching increased expression of the non-tenocyte-related genes, LPL, Sox-9 and Runx-2, while 4% stretching had minimal effects on the expression of these genes. In the in vivo study, moderate treadmill running (MTR) of aging mice (9 months) resulted in the increased proliferation rate of aging TSCs in culture, decreased lipid deposition, proteoglycan accumulation and calcification, and increased the expression of NS in the patellar tendons. These findings indicate that while aging impairs the proliferative ability of TSCs and reduces their stemness, moderate exercise can mitigate the deleterious effects of aging on TSCs and therefore may be responsible for decreased aging-induced tendon degeneration.     

Hypoxia enhances proliferation and stemness of human adipose-derived mesenchymal stem cells

The aim of the study was to obtain the highest number of multipotent adipose-derived mesenchymal stem cells (ADMSCs) by using culture conditions which favour cell expansion without loss of mesenchymal stem cells (MSC)-like properties. Based on the assumption that stem cells reside in niches characterized by hypoxic condition, we investigated if the low oxygen tension may improve the proliferation and stemness of ADMSCs. Intact adipose tissue was resected from eight subjects, and the stromal vascular fraction was obtained by using type II collagenase. The heterogeneity of cellular lineages was confirmed by immunophenotypic analysis that showed the presence of leukocytes (CD45+), endothelial cells (CD34+), and pericytes (CD140+). The immunophenotype of confluent ADMSCs was similar to that of bone marrow-derived MSCs, except for the expression of CD34, which was variable (donor-dependent) and inversely correlated to the CD36 expression. ADMSCs showed a high clonal efficiency (94.5 ± 1 %) and were able to generate osteoblastic, chondrocytic and adipocytic lineages. ADMSCs were cultured under normoxic (21 % O₂) and hypoxic (1 % O₂) conditions, and we found that hypoxia significantly favoured ADMSC proliferation and preserved the expression of stemness genes, i.e. Nanog and Sox2. Since hypoxia reflects the microenvironment in which ADMSCs must proliferate and differentiate, the culture in hypoxic condition allows to better understand the biology of these cells and their regenerative potential. Low oxygen concentrations promote cell proliferation and stemness, thus enriching the pool of cells potentially able to differentiate into multi-lineages, and extending the possibility of a long-term expansion.     

Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification

The ability of bovine blastocysts to recover after cryopreservation and thawing procedures is often assessed by evaluating their re-expansion during in vitro co-culture. However, the influence of factors such as feeder cell type and gas atmosphere on blastocyst survival and evolution have never been considered. This study therefore compared two cell co-culture systems and two different gas atmospheres to assess survival of in vitro produced bovine blastocysts after vitrification. Day-7 blastocysts (n=181) were vitrified in a mixture of 25% glycerol/25% ethylene glycol. After warming and dilution, they were co-cultured either on Buffalo rat liver cells (BRL CC cell line) or on granulosa cells (GR CC primary culture) in TCM 199 supplemented with 10% FCS and under an atmosphere of 5% or 20% O₂. Surviving and hatching rates were recorded at 24 h intervals for 3 days. After 72 h of culture, surviving blastocysts were treated for differential counting of inner cell mass (ICM) and trophectoderm cells. Blastocyst survival rates were higher when BRL and granulosa co-culture were performed under 20% oxygen as compared to 5% oxygen (20% O₂: 62% vs. 5% O₂: 25%, P<0.0001). However, the quality of blastocysts surviving in the granulosa co-culture condition was lower under 20% O₂ than under 5% O₂ as indicated by lower total and trophectoderm cell numbers (respectively 79±6 and 56±6 at 20% O₂ vs. 100±10 and 74±10 at 5% O₂, P<0.05), by an altered ICM/trophectoderm ratio (20% O₂: 28% vs. 5% O₂: 23%, P<0.05), by a higher total nuclear fragmentation (20% O₂: 3.7% vs. 5% O₂: 1.5%, P<0.05) and a trend to decreased hatching (20% O₂: 32% vs. 5% O₂: 81%, P=0.07). Whereas, for BRL co-culture, 20% O₂ yielded higher quality blastocysts than 5% O₂ as evaluated by higher ICM and trophectoderm cell numbers (19±1 and 71±5 at 20% O₂ vs. 15±2 and 48±9 at 5% O₂, respectively, P<0.05), by lower nuclear fragmentation in the ICM (20% O₂: 2.2% vs. 5% O₂: 6.7%, P<0.05). In conclusion, co-culture conditions may influence blastocysts survival and quality after cryopreservation. In our conditions, co-culture with BRL cells under 20% O₂ seems to be the best combination to evaluate blastocyst survival and quality after vitrification.     

High Oxygen Condition Facilitates the Differentiation of Mouse and Human Pluripotent Stem Cells into Pancreatic Progenitors and Insulin-producing Cells

Pluripotent stem cells have potential applications in regenerative medicine for diabetes. Differentiation of stem cells into insulin-producing cells has been achieved using various protocols. However, both the efficiency of the method and potency of differentiated cells are insufficient. Oxygen tension, the partial pressure of oxygen, has been shown to regulate the embryonic development of several organs, including pancreatic β-cells. In this study, we tried to establish an effective method for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells by culturing under high oxygen (O₂) conditions. Treatment with a high O₂ condition in the early stage of differentiation increased insulin-positive cells at the terminus of differentiation. We found that a high O₂ condition repressed Notch-dependent gene Hes1 expression and increased Ngn3 expression at the stage of pancreatic progenitors. This effect was caused by inhibition of hypoxia-inducible factor-1α protein level. Moreover, a high O₂ condition activated Wnt signaling. Optimal stage-specific treatment with a high O₂ condition resulted in a significant increase in insulin production in both mouse embryonic stem cells and human iPSCs and yielded populations containing up to 10% C-peptide-positive cells in human iPSCs. These results suggest that culturing in a high O₂ condition at a specific stage is useful for the efficient generation of insulin-producing cells.     

Design of a stirred multiwell bioreactor for expansion of CD34+ umbilical cord blood cells in hypoxic conditions

Besides having a metabolic role, oxygen is recognized as an important signaling stimulus for stem cells. In hematopoiesis, hypoxia seems to favor stem cell self‐renewal. In fact, long‐term repopulating hematopoietic stem cells reside in bone marrow at concentrations as low as 1% oxygen. However, O₂ concentration is difficult to control in vitro. Thermodynamically, we found significant differences between O₂ solubility in different media, and in presence of serum. Furthermore, we verified that medium equilibration with a hypoxic atmosphere requires several hours. Thus, in a static culture, the effective O₂ concentration in the cell immediate microenvironment is difficult to control and subject to concentration gradients. Stirred systems improve homogeneity within the culture volume. In this work, we developed a stirred bioreactor to investigate hypoxia effect on the expression of stem cell markers in CD34⁺ cells from umbilical cord blood. The stirring system was designed on top of a standard six‐well plate to favor continuity with conventional static conditions and transfer of culture protocols. The bioreactor volume (10 mL/well) is suitable for cell expansion and multiparametric flow cytometry analyses. First, it was tested at 21% O₂ for biocompatibility and other possible effects on the cells compared to static conditions. Then, it was used to study c‐kit expression of CD34⁺ cells at 5% O₂, using 21%‐O₂ cultures as a control. In hypoxia we found that CD34⁺ cells maintained a higher expression of c‐kit. Further investigation is needed to explore the dynamics of interaction between oxygen‐ and c‐kit‐dependent pathways at the molecular level. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Hypoxic culture enhances the expansion of rat bone marrow-derived mesenchymal stem cells via the regulatory pathways of cell division and apoptosis

This study aimed to examine the proliferative behavior and molecular mechanisms of rat bone marrow-derived MSCs (rBMSCs) cultured under three different oxygen concentrations. Passaged rBMSCs exhibited significantly greater proliferation rates at 1% O₂ and 5% O₂ than those at 18% O₂ and the cells exposed to 1% O₂ showed the highest proliferative potential, which was evidenced by the growth curves, colony-forming efficiencies, and CCK-8 absorbance values. The rBMSCs grown under hypoxic culture conditions (1% O₂ and 5% O₂) had the increased percentage of cells in S?+?G₂/M-phase and the decreased apoptotic index, compared with normoxia (18% O₂). It was revealed for the first time that there were more phosphohistone H3 (PHH3)-positive cells and higher expressions of proliferating cell nuclear antigen (PCNA) in the hypoxic cultures of rBMSCs than in the normoxic culture. Hypoxia upregulated the anti-apoptotic protein Bcl-2 and downregulated the pro-apoptotic proteins Bax and the cleaved caspase-3 in cultured rBMSCs. The levels of hypoxia-inducible factor-1α (HIF-1α) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) were increased in the hypoxic-cultured rBMSCs. Nevertheless, no significant difference was observed in p53 level of rBMSCs between different oxygen concentrations. In conclusion, the hypoxia exerts a promoting effect on the in vitro expansion of rBMSCs via several signaling and molecular pathways involved in the control of cell cycle and apoptosis.     

Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

The human THP-1 cell line is widely used as an in vitro model system for studying macrophage differentiation and function. Conventional culture conditions for these cells consist of ambient oxygen pressure (∼20% v/v) and medium supplemented with the thiol 2-mercaptoethanol (2-ME) and serum. In consideration of the redox activities of O₂ and 2-ME, and the extensive experimental evidence supporting a role for reactive oxygen species (ROS) in the differentiation and function of macrophages, we addressed the question of whether culturing THP-1 cells under a more physiologically relevant oxygen tension (5% O₂) in the absence of 2-ME and serum would alter THP-1 cell physiology. Comparisons of cultures maintained in 18% O₂ versus 5% O₂ indicated that reducing oxygen tension had no effect on the proliferation of undifferentiated THP-1 cells. However, decreasing the oxygen tension to 5% O₂ significantly increased the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. Removal of both 2-ME and serum from the medium decreased the proliferation of undifferentiated THP-1 cells but increased metabolic activity and the rate of differentiation under either oxygen tension. In differentiated THP-1 cells, lowering the oxygen tension to 5% O₂ decreased phagocytic activity, the constitutive release of β-hexosaminidase and LPS-induced NF-κB activation but enhanced LPS-stimulated release of cytokines. Collectively, these data demonstrate that oxygen tension influences THP-1 cell differentiation and primary macrophage functions, and suggest that culturing these cells under tightly regulated oxygen tension in the absence of exogenous reducing agent and serum is likely to provide a physiologically relevant baseline from which to study the role of the local redox environment in regulating THP-1 cell physiology.     

Human Adipose-Tissue Derived Stromal Cells in Combination with Hypoxia Effectively Support Ex Vivo Expansion of Cord Blood Haematopoietic Progenitors

The optimisation of haematopoietic stem and progenitor cell expansion is on demand in modern cell therapy. In this work, haematopoietic stem/progenitor cells (HSPCs) have been selected from unmanipulated cord blood mononuclear cells (cbMNCs) due to adhesion to human adipose-tissue derived stromal cells (ASCs) under standard (20%) and tissue-related (5%) oxygen. ASCs efficiently maintained viability and supported further HSPC expansion at 20% and 5% O₂. During co-culture with ASCs, a new floating population of differently committed HSPCs (HSPCs-1) grew. This suspension was enriched with СD34⁺ cells up to 6 (20% O₂) and 8 (5% O₂) times. Functional analysis of HSPCs-1 revealed cobble-stone area forming cells (CAFCs) and lineage-restricted colony-forming cells (CFCs). The number of CFCs was 1.6 times higher at tissue-related O₂, than in standard cultivation (20% O₂). This increase was related to a rise in the number of multipotent precursors - BFU-E, CFU-GEMM and CFU-GM. These changes were at least partly ensured by the increased concentration of MCP-1 and IL-8 at 5% O₂. In summary, our data demonstrated that human ASCs enables the selection of functionally active HSPCs from unfractionated cbMNCs, the further expansion of which without exogenous cytokines provides enrichment with CD34⁺ cells. ASCs efficiently support the viability and proliferation of cord blood haematopoietic progenitors of different commitment at standard and tissue-related O₂ levels at the expense of direct and paracrine cell-to-cell interactions.     

Growth of aortic vascular smooth muscle cells in lowered oxygen tension

Summary Primary cultures of vascular smooth muscle cells, isolated from rat aorta, were grown under normoxic (20% O₂) and mildly hypoxic (5 % O₂) conditions. Cells from both conditions were compared for growth characteristics, morphology, protein synthesis, lysosomal enzyme activity, and oxygen consumption. In no case was a consistently significant difference observed. These observations indicate that these cells can adapt or are adapted to mildly hypoxic conditions. Moreover, these results may indicate that the culture of vascular smooth muscle cells in mild hypoxia represents a closer approximation of in vivo growth conditions for these cells.Supported by HL19242     

Murine Embryonic Stem Cell Plasticity Is Regulated through Klf5 and Maintained by Metalloproteinase MMP1 and Hypoxia

Mouse embryonic stem cells (mESCs) are expanded and maintained pluripotent in vitro in the presence of leukemia inhibitory factor (LIF), an IL6 cytokine family member which displays pleiotropic functions, depending on both cell maturity and cell type. LIF withdrawal leads to heterogeneous differentiation of mESCs with a proportion of the differentiated cells apoptosising. During LIF withdrawal, cells sequentially enter a reversible and irreversible phase of differentiation during which LIF addition induces different effects. However the regulators and effectors of LIF–mediated reprogramming are poorly understood. By employing a LIF-dependent ‘plasticity’ test, that we set up, we show that Klf5, but not JunB is a key LIF effector. Furthermore PI3K signaling, required for the maintenance of mESC pluripotency, has no effect on mESC plasticity while displaying a major role in committed cells by stimulating expression of the mesodermal marker Brachyury at the expense of endoderm and neuroectoderm lineage markers. We also show that the MMP1 metalloproteinase, which can replace LIF for maintenance of pluripotency, mimics LIF in the plasticity window, but less efficiently. Finally, we demonstrate that mESCs maintain plasticity and pluripotency potentials in vitro under hypoxic/physioxic growth conditions at 3% O₂ despite lower levels of Pluri and Master gene expression in comparison to 20% O₂.     

Hypoxia/hypercapnia prevents iron-dependent cold injuries in cord blood stem and progenitor cells

BackgroundCold-induced cell injuries are associated with an increase in the cellular labile iron pool (LIP) followed by lipid peroxidation and alteration of mitochondrial function, which lead to cell death. Recently, we showed that incubation in a hypoxic/hypercapnic (HH) gas mixture improved the survival of a population of cord blood hematopoietic progenitors and CD34⁺ hematopoietic progenitor and stem cells in severe hypothermia. To explain the underlying mechanism, here we test if this HH-induced cytoprotection in cold conditions is associated with the level of LIP and lysosome stability.MethodsCord blood CD34⁺ cells were incubated in air (20% O₂/0.05% CO₂) or in the hypoxic (5% O₂)/hypercapnic (9% CO₂) atmosphere for 7days at 4°C and analyzed.ResultsIncubation in HH condition maintained the day 0 (D-0) level of LIP detected using a bleomycin-dependent method. This was associated with preservation of lysosome integrity and a higher cell survival. Conversely, in the air condition LIP was significantly increased. Also, the presence of a moderate concentration of iron chelator deferoximine improves the conservation of total CD34⁺ cells and committed progenitors in air condition. Pre-treatment of CD34⁺ cells with the lysomotropic agent imidazole induces significant decrease in the lysosomal stability and in all conditions. This is associated with an important decrease of survival of conserved cells and an increase in the cellular LIP level.DiscussionOur study showed that HH gas mixture cytoprotection during hypothermia maintains lysosome stability, which enables preservation of the cellular chelatable iron in the physiological ranges. These findings suggest a way to optimize cell conservation without freezing.