Low oxygen tension modulates the osteogenic differentiation of mouse embryonic stem cells

An, Seong Yeong,Heo, Jung Sun Elsevier 2018 Tissue & cell Vol.52 No.-

<P><B>Abstract</B></P> <P>This study examined the effects of low oxygen tension on the osteogenic differentiation of embryonic stem cells (ESCs) in a three-dimensional culture system. The high expression levels of hypoxia-related proteins hypoxia-inducible factor-1α and vascular endothelial growth factor were first validated in ESCs subjected to hypoxic conditions compared with normoxic controls. The osteogenic differentiation of hypoxic ESCs with either osteogenic or osteogenic factor-free media was subsequently evaluated by measuring alkaline phosphatase activity, intracellular calcium levels, matrix mineralization, and the protein levels of osteogenic markers Runt-related transcription factor 2 and osterix. We confirmed that hypoxia significantly stimulated ESC osteogenic activity; the strongest stimulation of ESC osteogenesis was exerted when cells were grown in osteogenic media. To identify differentially expressed genes associated with hypoxia-induced ESC differentiation, we performed microarray analysis of ESCs cultured in osteogenic media under normoxic and hypoxic conditions. This study demonstrated that differences in oxygen tension induced the differential expression of genes known to play roles in such processes as skeletal system development and signaling pathways for bone morphogenetic protein, Wnt, Notch, mitogen-activated protein kinase, and integrin. These findings reveal the effects of low oxygen tension on osteogenic progression in ESCs and provide insight into the molecular pathways that regulate ESC differentiation following exposure to hypoxia.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Effect of low oxygen tension on the osteogenic differentiation of embryonic stem cells in a 3D-culture system is provided. </LI> <LI> Differentially expressed genes associated with hypoxia-induced ESC differentiation are identified using microarray profiling. </LI> <LI> Molecular pathways which play roles in skeletal system development are determined. </LI> </UL> </P>

Hypoxia Mimicking Compounds Induce Chondrogenic Differentiation of Human Clonal Mesenchymal Stem Cells (hcMSCs) Via Hypoxia-Inducing Factor 1α (HIF-1α)

( A Rum Seo ),( Mi Hyun Lim ),( Kil Hwan Kim ),( Ok Hee Chang ),( Sun Uk Song ),( So Ra Park ),( Byoung Hyun Min ),( Byung Hyune Choi ) 한국조직공학·재생의학회 2011 조직공학과 재생의학 Vol.8 No.2

Hypoxia is known to play an important role in the cartilage development and normal function of adult cartilage. However, the effect and mechanism of hypoxia on the chondrogenesis of stem cells in vitro are not well understood yet. This study utilized hypoxia mimicking compounds of CoCl2 and desferrioxamine (DFX) to differentiate a clonal line of human mesenchymal stem cells (MSCs). Human clonal MSCs (hcMSCs) were cultured 3 dimensionally (3D) in alginate or fibrin hydrogel and differentiated into chondrogenic lineages for 7 or 14 days in the presence of 5 μM CoCl2 (or DFX) and/or transforming growth factor- β3 (TGF- β3). In RT-PCR analysis, both CoCl2 and DFX induced the expression of chondrogenic markers such as type II collagen and aggrecan as efficiently as TGF- β3. Co-treatment of CoCl2 (or DFX) and TGF- β3 did not show clear additive effect. Safranin-O staining of samples in fibrin hydrogel showed that CoCl2 also increased lacunae formation and accumulation of sulfated glycosaminoglycans (GAGs). Unexpectedly, both CoCl2 and TGF- β3 increased the protein level of HIF-1α. However, the expression of VEGF, a HIF-1 target gene, was induced only by CoCl2 but not by TGF- β3. Besides, HIF-1α·siRNA decreased specifically the expression of type II collagen and aggrecan induced by CoCl2 but not those induced by TGF- β3. These results suggest that the hypoxia mimicking compounds could be a useful tool to induce chondrogenic differentiation and cartilage tissue formation of MSCs, and probably utilize different mechanism from that of TGF-β.

Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation

Kim, Ji Hye,Yoon, Sei Mee,Song, Sun U.,Park, Sang Gyu,Kim, Won-Serk,Park, In Guk,Lee, Jinu,Sung, Jong-Hyuk MDPI 2016 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.17 No.9

<P>Hypoxia has diverse stimulatory effects on human adipose-derived stem cells (ASCs). In the present study, we investigated whether hypoxic culture conditions (2% O<SUB>2</SUB>) suppress spontaneous mineralization and osteogenic differentiation of ASCs. We also investigated signaling pathways and molecular mechanisms involved in this process. We found that hypoxia suppressed spontaneous mineralization and osteogenic differentiation of ASCs, and up-regulated mRNA and protein expression of Insulin-like growth factor binding proteins (IGFBPs) in ASCs. Although treatment with recombinant IGFBPs did not affect osteogenic differentiation of ASCs, siRNA-mediated inhibition of IGFBP3 attenuated hypoxia-suppressed osteogenic differentiation of ASCs. In contrast, overexpression of IGFBP3 via lentiviral vectors inhibited ASC osteogenic differentiation. These results indicate that hypoxia suppresses spontaneous mineralization and osteogenic differentiation of ASCs via intracellular IGFBP3 up-regulation. We determined that reactive oxygen species (ROS) generation followed by activation of the MAPK and PI3K/Akt pathways play pivotal roles in IGFBP3 expression under hypoxia. For example, ROS scavengers and inhibitors for MAPK and PI3K/Akt pathways attenuated the hypoxia-induced IGFBP3 expression. Inhibition of Elk1 and NF-κB through siRNA transfection also led to down-regulation of IGFBP3 mRNA expression. We next addressed the proliferative potential of ASCs with overexpressed IGFBP3, but IGFBP3 overexpression reduced the proliferation of ASCs. In addition, hypoxia reduced the osteogenic differentiation of bone marrow-derived clonal mesenchymal stem cells. Collectively, our results indicate that hypoxia suppresses the osteogenic differentiation of mesenchymal stem cells via IGFBP3 up-regulation.</P>

Conversion to Veno-arteriovenous Extracorporeal Membrane Oxygenation for Differential Hypoxia

Cha Ho Jeong,Kim Jong Woo,Kang Dong Hoon,Moon Seong Ho,Kim Sung Hwan,Jung Jae Jun,Yang Jun Ho,Byun Joung Hun 대한심장혈관흉부외과학회 2023 Journal of Chest Surgery (J Chest Surg) Vol.56 No.4

Background: Patients who require initial venoarterial extracorporeal membrane oxygenation (VA ECMO) support may need to undergo veno-arteriovenous ECMO (VAV ECMO) conversion. However, there are no definitive criteria for conversion to VAV ECMO. We report 9 cases of VAV ECMO at Gyeongsang National University Changwon Hospital, Gyeongsang National University College of Medicine. Methods: Of 158 patients who received ECMO support between January 2017 and June 2019, 82 were supported by initial VA ECMO. We retrospectively reviewed the medical records of 9 patients (7 men and 2 women; age, 53.1±19.4 years) who had differential hypoxia and required VAV ECMO support. Percutaneous transaortic catheter venting was used to detect the differential hypoxia. Results: Among the 82 patients who received VA ECMO support, 9 (10.9%) had differential hypoxia and required conversion to VAV ECMO support. The mean time from VA ECMO support to VAV ECMO support and the mean duration of the VAV support were 2.1±2.2 days and 1.9±1.5 days, respectively. The average peak inspiratory pressure before and after VAV ECMO application was 23.89±3.95 cmH2O and 20.67±5.72 cmH2O, respectively, decreasing by an average of 3.2±3.5 cmH2O (p=0.040). The PaO2/FiO2 ratio was kept below 100 mm Hg in survivors and non-survivors for 116±65.4 and 250±124.9 minutes, respectively (p=0.016). Six patients underwent extracorporeal cardiopulmonary resuscitation, of whom 4 survived (67%). The overall survival rate of patients who underwent conversion from VA ECMO to VAV ECMO was approximately 56%. Conclusion: Rapid detection of differential hypoxia is required when VA ECMO is applied, and efficient conversion to VAV ECMO may be critical for patient survival.

Biochemical and Morphological Effects of Hypoxic Environment on Human Embryonic Stem Cells in Long-Term Culture and Differentiating Embryoid Bodies

Hee-Joung Lim,한지유,Dong-Hun Woo,Sung-Eun Kim,김슬기,강희규,김종훈 한국분자세포생물학회 2011 Molecules and cells Vol.31 No.2

The mammalian reproductive tract is known to contain 1.5-5.3% oxygen (O_2), but human embryonic stem cells (hESCs) derived from preimplantation embryos are typically cultured under 21% O_2 tension. The aim of this study was to investigate the effects of O_2 tension on the long-term culture of hESCs and on cell-fate determination during early differentiation. hESCs and embryoid bodies (EBs) were grown under different O2 tensions (3, 12, and 21% O_2). The expression of markers associated with pluripotency, embryonic germ layers, and hypoxia was analyzed using RT-PCR, immunostaining, and Western blotting. Proliferation, apoptosis, and chromosomal aberrations were examined using BrdU incorporation, caspase-3 immunostaining, and karyotype analysis, respectively. Structural and morphological changes of EBs under different O_2 tensions were comparatively examined using azan- and hematoxylin-eosin staining, and scanning and transmission electron microscopy. Mild hypoxia (12% O_2) increased the number of cells expressing Oct4/Nanog and reduced BrdU incorporation and aneuploidy. The percentage of cells positive for active caspase-3, which was high during normoxia (21% O_2), gradually decreased when hESCs were continuously cultured under mild hypoxia. EBs subjected to hypoxia (3% O_2) exhibited well-differentiated microvilli on their surface, secreted high levels of collagen, and showed enhanced differentiation into primitive endoderm. These changes were associated with increased expression of Foxa2, Sox17, AFP, and GATA4 on the EB periphery. Our data suggest that mild hypoxia facilitates the slow mitotic division of hESCs in long-term culture and reduces the frequency of chromosomal abnormalities and apoptosis. In addition, hypoxia promotes the differentiation of EBs into extraembryonic endoderm.

Microarray Analysis of the Hypoxia-induced Gene Expression Profile in Malignant C6 Glioma Cells

Huang, Xiao-Dong,Wang, Ze-Fen,Dai, Li-Ming,Li, Zhi-Qiang Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.9

Hypoxia is commonly featured during glioma growth and plays an important role in the processes underlying tumor progression to increasing malignancy. Here we compared the gene expression profiles of rat C6 malignant glioma cells under normoxic and hypoxic conditions by cDNA microarray analysis. Compared to normoxic culture conditions, 180 genes were up-regulated and 67 genes were down-regulated under hypoxia mimicked by $CoCl_2$ treatment. These differentially expressed genes were involved in mutiple biological functions including development and differentiation, immune and stress response, metabolic process, and cellular physiological response. It was found that hypoxia significantly regulated genes involved in regulation of glycolysis and cell differentiation, as well as intracellular signalling pathways related to Notch and focal adhesion, which are closely associated with tumor malignant growth. These results should facilitate investigation of the role of hypoxia in the glioma development and exploration of therapeutic targets for inhibition of glioma growth.

Effects of CoCl₂ on multi-lineage differentiation of C3H/10T1/2 mesenchymal stem cells

Hong Il Yoo,Yeon Hee Moon,Min Seok Kim 대한생리학회-대한약리학회 2016 The Korean Journal of Physiology & Pharmacology Vol.20 No.1

Mesenchymal stem cells (MSCs) in the bone marrow and other somatic tissues reside in an environment with relative low oxygen tension. Cobalt chloride (CoCl₂) can mimic hypoxic conditions through transcriptional changes of some genes including hypoxia-inducible factor-1a (HIF-1a) and vascular endothelial growth factor (VEGF). This study evaluated the potential role of CoCl₂ preconditioning on multi-lineage differentiation of C3H/10T1/2, a murine MSC line to understand its possible molecular mechanisms <i>in vitro</i>. CoCl₂ treatment of MSCs markedly increased HIF-1a and VEGF mRNA, and protein expression of HIF-1a. Temporary preconditioning of MSCs with CoCl₂ induced up-regulation of osteogenic markers including alkaline phosphatase, osteocalcin, and type I collagen during osteogenic differentiation, followed by enhanced mineralization. CoCl₂ also increased chondrogenic markers including aggrecan, sox9, and type II collagen, and promoted chondrocyte differentiation. CoCl₂ suppressed the expression of adipogenic markers including PPARγ, aP2, and C/EBPa, and inhibited adipogenesis. Temporary preconditioning with CoCl₂ could affect the multi-lineage differentiation of MSCs.

Effects of CoCl2 on multi-lineage differentiation of C3H/10T1/2 mesenchymal stem cells

유홍일,문연희,김민석 대한약리학회 2016 The Korean Journal of Physiology & Pharmacology Vol.20 No.1

Mesenchymal stem cells (MSCs) in the bone marrow and other somatic tissues reside in an environment with relative low oxygen tension. Cobalt chloride (CoCl2) can mimic hypoxic conditions through transcriptional changes of some genes including hypoxia-inducible factor-1a (HIF-1a) and vascular endothelial growth factor (VEGF). This study evaluated the potential role of CoCl2 preconditioning on multi-lineage differentiation of C3H/10T1/2, a murine MSC line to understand its possible molecular mechanisms in vitro. CoCl2 treatment of MSCs markedly increased HIF-1a and VEGF mRNA, and protein expression of HIF-1a. Temporary preconditioning of MSCs with CoCl2 induced up-regulation of osteogenic markers including alkaline phosphatase, osteocalcin, and type I collagen during osteogenic differentiation, followed by enhanced mineralization. CoCl2 also increased chondrogenic markers including aggrecan, sox9, and type II collagen, and promoted chondrocyte differentiation. CoCl2 suppressed the expression of adipogenic markers including PPARγ, aP2, and C/EBPa, and inhibited adipogenesis. Temporary preconditioning with CoCl2 could affect the multi-lineage differentiation of MSCs.

Effects of CoCl₂ on multi-lineage differentiation of C3H/10T1/2 mesenchymal stem cells

Yoo, Hong Il,Moon, Yeon Hee,Kim, Min Seok The Korean Society of Pharmacology 2016 The Korean Journal of Physiology & Pharmacology Vol.20 No.1

Mesenchymal stem cells (MSCs) in the bone marrow and other somatic tissues reside in an environment with relative low oxygen tension. Cobalt chloride ($CoCl_2$) can mimic hypoxic conditions through transcriptional changes of some genes including hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) and vascular endothelial growth factor (VEGF). This study evaluated the potential role of $CoCl_2$ preconditioning on multi-lineage differentiation of C3H/10T1/2, a murine MSC line to understand its possible molecular mechanisms in vitro. $CoCl_2$ treatment of MSCs markedly increased HIF-$1{\alpha}$ and VEGF mRNA, and protein expression of HIF-$1{\alpha}$. Temporary preconditioning of MSCs with $CoCl_2$ induced up-regulation of osteogenic markers including alkaline phosphatase, osteocalcin, and type I collagen during osteogenic differentiation, followed by enhanced mineralization. $CoCl_2$ also increased chondrogenic markers including aggrecan, sox9, and type II collagen, and promoted chondrocyte differentiation. $CoCl_2$ suppressed the expression of adipogenic markers including $PPAR{\gamma}$, aP2, and $C/EBP{\alpha}$, and inhibited adipogenesis. Temporary preconditioning with $CoCl_2$ could affect the multi-lineage differentiation of MSCs.

Enhanced Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells by Hypoxia-induced Chemicals

Hyoungki KIM,Soonjo KWON 한국생물공학회 2020 한국생물공학회 학술대회 Vol.2020 No.10