Low oxygen tension potentiates proliferation and stemness but not multilineage differentiation of caprine male germline stem cells

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O₂) environment, whereas, their in-vitro expansion is typically performed under normoxia (20–21% O₂). The comparative information about the effects of low and normal O₂ levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O₂) and normoxia (21% O₂). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p?<?0.05) improved, thus, yielding a significantly (p?<?0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O₂ conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p?<?0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

     

Mitogenic action of hypoxia upon cutaneous neuroepithelial cells in developing zebrafish

In zebrafish, cutaneous neuroepithelial cells (NECs) contain serotonin (5‐HT) and are believed to initiate physiological and behavioral responses to hypoxia during embryonic and early larval development, when mature gills and O₂ chemoreceptors are not yet present. The number of skin NECs rapidly declines as embryos develop into larvae, but acclimation to hypoxia leads to retention of a greater number of these cells. We hypothesized that reduction of the partial pressure of oxygen (P _O2) in water would stimulate mitosis in cutaneous NECs in zebrafish. Zebrafish were exposed to 5‐bromo‐2′‐deoxyuridine (BrdU) and immunolabeled with antibodies against serotonin and BrdU to identify mitotic skin cells, including NECs. Cells were imaged and quantified using confocal microscopy. From embryonic to larval stages, we observed an overall increase in the number of BrdU‐positive cells in the skin, but a decrease in BrdU‐positive serotonergic NECs. Exposure of larvae to hypoxia (P _O2 = 30 mmHg) in vivo for 24 h produced a 1.7‐fold increase in the number of NECs labeled with BrdU. We conclude that under normal environmental P _O2 the population of cutaneous NECs declines due to a decrease in mitotic activity. During environmental hypoxia, the number of NECs undergoing cell division in the skin is increased, and this promotes retention of NECs under these conditions. These data demonstrate the direct action of hypoxia upon the cell cycle of cutaneous NECs in developing zebrafish, and support the notion that cutaneous NECs are embryonic O₂ chemoreceptors. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 789–801, 2017     

Electrical stimulation of human embryonic stem cells: Cardiac differentiation and the generation of reactive oxygen species

Exogenous electric fields have been implied in cardiac differentiation of mouse embryonic stem cells and the generation of reactive oxygen species (ROS). In this work, we explored the effects of electrical field stimulation on ROS generation and cardiogenesis in embryoid bodies (EBs) derived from human embryonic stem cells (hESC, line H13), using a custom-built electrical stimulation bioreactor. Electrical properties of the bioreactor system were characterized by electrochemical impedance spectroscopy (EIS) and analysis of electrical currents. The effects of the electrode material (stainless steel, titanium-nitride-coated titanium, titanium), length of stimulus (1 and 90 s) and age of EBs at the onset of electrical stimulation (4 and 8 days) were investigated with respect to ROS generation. The amplitude of the applied electrical field was 1 V/mm. The highest rate of ROS generation was observed for stainless steel electrodes, for signal duration of 90 s and for 4-day-old EBs. Notably, comparable ROS generation was achieved by incubation of EBs with 1 nM H₂O₂. Cardiac differentiation in these EBs was evidenced by spontaneous contractions, expression of troponin T and its sarcomeric organization. These results imply that electrical stimulation plays a role in cardiac differentiation of hESCs, through mechanisms associated with the intracellular generation of ROS.     

Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells

The study of how human embryonic stem cells (hESCs) differentiate into insulin-producing beta cells has twofold significance: first, it provides an in vitro model system for the study of human pancreatic development, and second, it serves as a platform for the ultimate production of beta cells for transplantation into patients with diabetes. The delineation of growth factor interactions regulating pancreas specification from hESCs in vitro is critical to achieving these goals. In this study, we describe the roles of growth factors bFGF, BMP4 and Activin A in early hESC fate determination. The entire differentiation process is carried out in serum-free chemically-defined media (CDM) and results in reliable and robust induction of pancreatic endoderm cells, marked by PDX1, and cell clusters co-expressing markers characteristic of beta cells, including PDX1 and insulin/C-peptide. Varying the combinations of growth factors, we found that treatment of hESCs with bFGF, Activin A and BMP4 (FAB) together for 3–4 days resulted in strong induction of primitive-streak and definitive endoderm-associated genes, including MIXL1, GSC, SOX17 and FOXA2. Early proliferative foregut endoderm and pancreatic lineage cells marked by PDX1, FOXA2 and SOX9 expression are specified in EBs made from FAB-treated hESCs, but not from Activin A alone treated cells. Our results suggest that important tissue interactions occur in EB-based suspension culture that contribute to the complete induction of definitive endoderm and pancreas progenitors. Further differentiation occurs after EBs are embedded in Matrigel and cultured in serum-free media containing insulin, transferrin, selenium, FGF7, nicotinamide, islet neogenesis associated peptide (INGAP) and exendin-4, a long acting GLP-1 agonist. 21–28 days after embedding, PDX1 gene expression levels are comparable to those of human islets used for transplantation, and many PDX1⁺ clusters are formed. Almost all cells in PDX1⁺ clusters co-express FOXA2, HNF1ß, HNF6 and SOX9 proteins, and many cells also express CPA1, NKX6.1 and PTF1a. If cells are then switched to medium containing B27 and nicotinamide for 7–14 days, then the number of insulin⁺ cells increases markedly. Our study identifies a new chemically defined culture protocol for inducing endoderm- and pancreas-committed cells from hESCs and reveals an interplay between FGF, Activin A and BMP signaling in early hESC fate determination.     

Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm

Nodal, a member of the TGF-β family of signaling molecules, has been implicated in pluripotency in human embryonic stem cells (hESCs) [Vallier, L., Reynolds, D., Pedersen, R.A., 2004a. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev. Biol. 275, 403-421], a finding that seems paradoxical given Nodal's central role in mesoderm/endoderm specification during gastrulation. In this study, we sought to clarify the role of Nodal signaling during hESC differentiation by constitutive overexpression of the endogenous Nodal inhibitors Lefty2 (Lefty) and truncated Cerberus (Cerb-S) and by pharmacological interference using the Nodal receptor antagonist SB431542. Compared to wildtype (WT) controls, embryoid bodies (EBs) derived from either Lefty or Cerb-S overexpressing hESCs showed increased expression of neuroectoderm markers Sox1, Sox3, and Nestin. Conversely, they were negative for a definitive endoderm marker (Sox17) and did not generate beating cardiomyocyte structures in conditions that allowed mesendoderm differentiation from WT hESCs. EBs derived from either Lefty or Cerb-S expressing hESCs also contained a greater abundance of neural rosette structures as compared to controls. Differentiating EBs derived from Lefty expressing hESCs generated a dense network of β-tubulin III positive neurites, and when Lefty expressing hESCs were grown as a monolayer and allowed to differentiate, they generated significantly higher numbers of β-tubulin positive neurons as compared to wildtype hESCs. SB431542 treatments reproduced the neuralising effects of Lefty overexpression in hESCs. These results show that inhibition of Nodal signaling promotes neuronal specification, indicating a role for this pathway in controlling early neural development of pluripotent cells.     

Inclusive estimation of complex antigen presentation functions of monocyte-derived dendritic cells differentiated under normoxia and hypoxia conditions

Dendritic cells (DCs) generated from monocytes under 20% O₂ are now used as therapeutic tools for cancer patients. However, the O₂ concentration is between 3 and 0.5% in most tissues. We evaluated these complicated functions of DCs under oxygen tensions mimicking in vivo situations. Immature DCs (imDCs) were generated from monocytes using IL-4 and GM-CSF under normoxia (20% O₂; N-imDCs) or hypoxia (1% O₂; H-imDCs). Mature DCs (mDCs) were induced with LPS. DCs were further exposed to normoxia (N/N-DCs) or hypoxia (N/H-DCs and H/H-DCs) conditions. Using a 2-D culture system, H-DCs were smaller in size than N-DCs, and H/H-DCs exhibited higher allo-T cell stimulation ability than N/N-DCs and N/H-DCs. On the other hand, motility and phagocytic ability of H/H-DCs were significantly lower than those of N/H-DCs and N/N-DCs. In a 3-D culture system, however, maturation of H/H-imDCs and N/H-imDCs was suppressed compared with N/N-imDCs as a result of their decreased motility and phagocytosis. Interestingly, silencing of HIF-1α by RNA interference decreased CD83 expression without affecting any antigen presentation abilities except for the ability to stimulate the allo-T cell population. Our data could help our understanding of DCs, especially therapeutic DCs, in vivo.     

Cluster characterization of mouse embryonic stem cell-derived pluripotent embryoid bodies in four distinct developmental stages

The formation of embryoid bodies (EBs) is the principal step in the differentiation of embryonic stem (ES) cells. In this study, the morphological characteristics and gene expression patterns of EBs related to the sequential stages of embryonic development were well defined in four distinct developmental groups over 112 days of culture: early-stage EBs groups (1–7 days of differentiation), mid-stage EBs groups (9–15 days of differentiation), maturing EBs groups (17–45 days of differentiation) and matured EBs groups (50 days of differentiation). We first determined definite histological location of apoptosis within EBs and the sequential expression of molecular markers representing stem cells (Oct4, SSEA-1, Sox-2 and AKP), germ cells (Fragilis, Dazl, c-kit, StellaR, Mvh and Stra8), ectoderm (Neurod, Nestin and Neurofilament), mesoderm (Gata-1, Flk-1 and Hbb) and endoderm (AFP and Transthyretin). Our results revealed that developing EBs possess either pluripotent stem cell or germ cell states and that three-dimensional aggregates of EBs initiate mES cell differentiation during prolonged culture in vitro. Therefore, we suggest that this EB system to some extent recapitulates the early developmental processes occurring in vivo.     

Activation and damage of endothelial cells upon hypoxia/reoxygenation. Effect of extracellular pH

Disturbances of blood flow upon vascular occlusions and spasms result in hypoxia and acidosis, while its subsequent restoration leads to reoxygenation and pH normalization (re-alkalization) in ischemic sites of the vascular bed. The effect of hypoxia/reoxygenation on activation and stimulation of apoptosis in cultured human endothelial cells was studied. The cells were subjected to hypoxia (2% O₂, 5% CO₂, 93% N₂) for 24 h followed by reoxygenation (21% O₂, 5% CO₂, 74% N₂) for 5 h. Reoxygenation was carried out at different pH-6.4 (preservation of acidosis after hypoxia), 7.0, and 7.4 (partial and complete re-alkalization, respectively). Hypoxia only slightly (by ～30%) increased the cell adhesion molecule ICAM-1 content on the cell surface, whereas reoxygenation more than doubled its expression. The reoxygenation effect depended on the medium acidity, and ICAM-1 increase was more pronounced at pH 7.0 compared to that at pH 6.4 and 7.4. Neither hypoxia nor reoxygenation induced expression of two other cell adhesion molecules, VCAM and E-selectin. Incubation of cells under hypoxic conditions but not reoxygenation stimulated secretion of von Willebrand factor and increased its concentration in the culture medium by more than 4 times. The percentage of cells containing apoptosis marker, activated caspase-3, was increased by approximately 1.5 times upon hypoxia as well as hypoxia/reoxygenation. Maximal values were achieved when reoxygenation was performed at pH 7.0. These data show that hypoxia/reoxygenation stimulate pro-inflammatory activation (ICAM-1 expression) and apoptosis (caspase-3 activation) of endothelial cells, and the extracellular pH influences both processes.     

The expression of p53-target genes in the hypoxia-tolerant subterranean mole-rat is hypoxia-dependent and similar to expression patterns in solid tumors

The tumor suppressor gene p53, in response to DNA damage/hypoxia, induces growth arrest and/or apoptosis. Inactivation of p53, by mutations and/or overexpression of the mdm2 gene, confers a selective advantage to tumor cells under hypoxic microenvironment during tumor progression. The mole rat, Spalax, spends its life underground at low-oxygen tensions and hence has developed a wide range of respiratory/molecular adaptations to hypoxic stress. We previously reported that the highly conserved p53 Arg(R)-174 is substituted by lysine (K) in Spalax, identical to a tumor- associated mutation. Functionality assays revealed that Spalax p53 and human R174K-mutated p53 were unable to induce human/Spalax apaf1, an apoptotic target gene, while over-activating the mdm2 gene. Moreover, cells transfected with human p53 underwent more extensive apoptosis (44.8%) as compared to Spalax p53 (23.2%) transfected cells. To support our hypothesis that the pattern of activity in Spalax is related to hypoxia-tolerance, we quantified apaf1 and mdm2 mRNA levels under normoxia (21% O₂), short-acute hypoxic stress (5 h at 6% O₂),and long-mild hypoxic insult (44 h at 10% O₂). Results were compared to those of rats under similar conditions. Following hypoxia, Spalax apaf1 mRNA levels decreased significantly, but increased in rats. apip mRNA levels, a negative regulator of apaf1, increased in Spalax and decreased in rats. mdm2 mRNA levels under hypoxia were significantly higher in Spalax. We conclude that, similar to our previous in-vitro work, two parallel hypoxia-adaptive mechanisms evolved in Spalax: mutated p53 and p53 response element leading to a bias against apoptosis and increased mdm2, which are analogous to observations in tumor development.     

人诱导多能干细胞与人胚胎干细胞分化过程中Oct4/Nanog基因表达的比较

目的：比较通过慢病毒方法获得的人诱导多能性干细胞（iPSCs）与人胚胎干细胞（hESCs）分化过程中全能性基因Oct4、Nanog的表达变化。方法：收集分化不同时间点的拟胚体（EBs）,检测三胚层分化基因以及全能性基因Oct4/Nanog的表达,并通过畸胎瘤组织切片的荧光染色分析Oct4的表达。结果：iPSCs获得的EB中内外三胚层分化基因表达的出现明显晚于hESCs来源的EB。不同于hESCs,iPSCs悬浮培养获得的EBs在体外培养18天未见内源性Oct4、Nanog基因表达的下调。未分化的iPSCs注射严重联合免疫缺陷（SCID）小鼠培养10周后获得的畸胎瘤中仍存在Oct4阳性的细胞,但iPS-#2中明显少于iPS-#5。结论：通过慢病毒方法获得的iPSCs虽然具有向三胚层分化的能力,但在分化过程中仍维持较高水平的全能性基因Oct4、Nanog的表达。     

Expression of estrogen receptor-alpha and -beta, glucocorticoid receptor, and progesterone receptor genes in human embryonic stem cells and embryoid bodies

Human embryonic stem cells (hESCs) have the potential to differentiate into various cell types, and the three germ layers in vivo and in vitro. They are therefore useful in transplantation and tissue engineering. Here, we describe the expression patterns of selected steroid receptor mRNAs - estrogen receptor-alpha (ER-alpha), ER-beta, glucocorticoid receptor (GR), and progesterone receptor (PR) - in undifferentiated hESCs and embryoid bodies (EBs) cultured for 2, 4, and 6 d, as assessed by real-time PCR, in order to define the possible influence of steroid hormones on the differentiation of hESCs. These receptor mRNAs were expressed in undifferentiated hESCs and EBs. The expression of PR mRNA only decreased during the differentiation of EBs but not of hESCs. Immunohistochemical analysis gave strong staining of ER-alpha, ER-beta, and GR proteins in the nuclei of hESCs and EBs, whereas PR was not detected. We also examined the potential of these steroid hormones to direct the differentiation of hESCs in vitro. The expression of 11 cell-specific markers representing 3 germ layers and 5 tissue types was used to assess the differentiation of hESCs. We found that certain endodermal marker genes were either only expressed in the estrogen-treated group or their expression was stimulated in that group, suggesting that steroid hormones can control the differentiation of hESCs into various cell types.     

Thermoregulatory and metabolic responses of Japanese quail to hypoxia

Common responses to hypoxia include decreased body temperature (T_b) and decreased energy metabolism. In this study, the effects of hypoxia and hypercapnia on T_b and metabolic oxygen consumption (V.O₂) were investigated in Japanese quail (Coturnix japonica). When exposed to hypoxia (15, 13, 11 and 9% O₂), T_b decreased only at 11% and 9% O₂ compared to normoxia; quail were better able to maintain T_b during acute hypoxia after a one-week acclimation to 10% O₂. V.O₂ also decreased during hypoxia, but at 9% O₂ this was partially offset by increased anaerobic metabolism. T_b and V.O₂ responses to 9% O₂ were exaggerated at lower ambient temperature (T_a), reflecting a decreased lower critical temperature during hypoxia. Conversely, hypoxia had little effect on T_b or V.O₂ at higher T_a (36 °C). We conclude that Japanese quail respond to hypoxia in much the same way as mammals, by reducing both T_b and V.O₂. No relationship was found between the magnitudes of decreases in T_b and V.O₂ during 9% O₂, however. Since metabolism is the source of heat generation, this suggests that Japanese quail increase thermolysis to reduce T_b. During hypercapnia (3, 6 and 9% CO₂), T_b was reduced only at 9% CO₂ while V.O₂ was unchanged.     

Acute hypoxia alters eicosanoid production of perfused pulmonary artery endothelial cells in culture

Hypoxia alters vascular tone which regulates regional blood flow in the pulmonary circulation. Endothelial derived eicosanoids alter vascular tone and blood flow and have been implicated as modulators of hypoxic pulmonary vasoconstriction. Eicosanoid production was measured in cultured bovine pulmonary endothelial cells during constant flow and pressure perfusion at two oxygen tensions (hypoxia: 4% O₂, 5% CO₂, 91% N₂; normoxia: 21% O₂, 5% CO₂, 74% N₂). Endothelial cells were grown to confluence on microcarrier beads. Cell cartridges (N=8) containing 2 ml of microcarrier beads ( 5 × 10⁶ cells) were constantly perfused (3 ml/min) with Krebs' solutions (pH 7.4, T 37°C) equilibrated with each gas mixture. After a ten minute equilibration period, lipids were extracted (C₁₈ Sep Pak®) from twenty minute aliquots of perfusate over three hours (nine aliquots per cartridge). Eicosanoids (6-keto PGF_1α; TXB₂; and total leukotriene [LT - LTC₄, LTD₄, LTE₄, LTF₄]) were assayed by radioimmunoassay. Eicosanoid production did not vary over time. 6-keto PGF_1α production was increased during hypoxia (normoxia 291 ± 27 vs hypoxia 395 ± 35 ng/min/gm protein; p < 0.01). Thromboxane production (normoxia 19 ± 2 vs hypoxia 20 ± 2 ng/min/gm protein) and total leukotriene production (normoxia 363 ± 35 vs hypoxia 329 ± 29 ng/min/gm protein) did not change with hypoxia. These data demonstrated that oxygen increased endothelial prostacyclin production but did not effect thromboxane or leukotriene production.     

Effect of Oxygen on Cardiac Differentiation in Mouse iPS Cells: Role of Hypoxia Inducible Factor-1 and Wnt/Beta-Catenin Signaling

Background

Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes.

Objective

We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS) cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha) and the canonical Wnt pathway in this process.

Methods

Embryoid bodies (EBs) derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture.

Results

At 14 days of differentiation, 59±2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway.

Conclusion

Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.     

Buoyancy control of four species of eleotrid fishes during aquatic surface respiration

Synopsis Four species of Australian Eleotridae from hypoxic habitats were examined in the laboratory to study buoyancy control in hypoxic water (<10 torr) when performing aquatic surface respiration (ASR; irrigating gills with upper millimeter of surface water). A conflict can arise here because O₂ can be reabsorbed from the swimbladder (reducing buoyancy) at a time when additional lift may be required to perform ASR. Three species were negatively buoyant and initially performed ASR while resting on the bottom in shallow water. After 24 h swimbladder lift increased to nearly neutral and ASR was performed while fish were pelagic. The fourth species remained pelagic at near neutral buoyancy in hypoxic water. With sudden exposure to hypoxia these physoclists reabsorbed between 5–27% (depending on species) of swimbladder volume (standard pressure) during the initial 30–90 min exposure to hypoxia. Additional experiments on one species (Hypseleotris galii) showed such loss to occur at O₂ tensions below 68 torr and when O₂ declined rapidly (2.17 torr min^-1). Secretion of gas compensated for losses under slower, natural rates of nocturnal O₂ decline. Eleotrids appear to reduce the conflict between respiration and buoyancy control in hypoxia by restricting gas reabsorbtion from the swimbladder and by rapidly secreting gases into the swimbladder.