1.Hypoxia promotes differentiation of human induced pluripotent stem cells into embryoid bodies in vitro.
Li Jun FANG ; Zi Bei FENG ; Jing Yi MEI ; Jia Hui ZHOU ; Zhan Yi LIN
Journal of Southern Medical University 2022;42(6):929-936
OBJECTIVE:
To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms.
METHODS:
EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment.
RESULTS:
The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of β-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, β-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05).
CONCLUSION
Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.
Echinomycin/metabolism*
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Embryoid Bodies/metabolism*
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Humans
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Hypoxia/metabolism*
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Induced Pluripotent Stem Cells/metabolism*
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beta Catenin/metabolism*
2.Intermittent compressive force induces cell cycling and reduces apoptosis in embryoid bodies of mouse induced pluripotent stem cells.
Jeeranan MANOKAWINCHOKE ; Phoonsuk LIMRAKSASIN ; Hiroko OKAWA ; Prasit PAVASANT ; Hiroshi EGUSA ; Thanaphum OSATHANON
International Journal of Oral Science 2022;14(1):1-1
In vitro manipulation of induced pluripotent stem cells (iPSCs) by environmental factors is of great interest for three-dimensional (3D) tissue/organ induction. The effects of mechanical force depend on many factors, including force and cell type. However, information on such effects in iPSCs is lacking. The aim of this study was to identify a molecular mechanism in iPSCs responding to intermittent compressive force (ICF) by analyzing the global gene expression profile. Embryoid bodies of mouse iPSCs, attached on a tissue culture plate in 3D form, were subjected to ICF in serum-free culture medium for 24 h. Gene ontology analyses for RNA sequencing data demonstrated that genes differentially regulated by ICF were mainly associated with metabolic processes, membrane and protein binding. Topology-based analysis demonstrated that ICF induced genes in cell cycle categories and downregulated genes associated with metabolic processes. The Kyoto Encyclopedia of Genes and Genomes database revealed differentially regulated genes related to the p53 signaling pathway and cell cycle. qPCR analysis demonstrated significant upregulation of Ccnd1, Cdk6 and Ccng1. Flow cytometry showed that ICF induced cell cycle and proliferation, while reducing the number of apoptotic cells. ICF also upregulated transforming growth factor β1 (Tgfb1) at both mRNA and protein levels, and pretreatment with a TGF-β inhibitor (SB431542) prior to ICF abolished ICF-induced Ccnd1 and Cdk6 expression. Taken together, these findings show that TGF-β signaling in iPSCs enhances proliferation and decreases apoptosis in response to ICF, that could give rise to an efficient protocol to manipulate iPSCs for organoid fabrication.
Animals
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Apoptosis
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Cell Cycle
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Cell Differentiation
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Embryoid Bodies
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Induced Pluripotent Stem Cells/metabolism*
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Mice
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Transforming Growth Factor beta/pharmacology*
3.Recapitulating cortical development with organoid culture in vitro and modeling abnormal spindle-like (ASPM related primary) microcephaly disease.
Rui LI ; Le SUN ; Ai FANG ; Peng LI ; Qian WU ; Xiaoqun WANG
Protein & Cell 2017;8(11):823-833
The development of a cerebral organoid culture in vitro offers an opportunity to generate human brain-like organs to investigate mechanisms of human disease that are specific to the neurogenesis of radial glial (RG) and outer radial glial (oRG) cells in the ventricular zone (VZ) and subventricular zone (SVZ) of the developing neocortex. Modeling neuronal progenitors and the organization that produces mature subcortical neuron subtypes during early stages of development is essential for studying human brain developmental diseases. Several previous efforts have shown to grow neural organoid in culture dishes successfully, however we demonstrate a new paradigm that recapitulates neocortical development process with VZ, OSVZ formation and the lamination organization of cortical layer structure. In addition, using patient-specific induced pluripotent stem cells (iPSCs) with dysfunction of the Aspm gene from a primary microcephaly patient, we demonstrate neurogenesis defects result in defective neuronal activity in patient organoids, suggesting a new strategy to study human developmental diseases in central nerve system.
Action Potentials
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physiology
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Biomarkers
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metabolism
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Cell Culture Techniques
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Embryoid Bodies
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cytology
;
metabolism
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Gene Expression
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Humans
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Induced Pluripotent Stem Cells
;
cytology
;
metabolism
;
Lateral Ventricles
;
cytology
;
growth & development
;
metabolism
;
Microcephaly
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genetics
;
metabolism
;
pathology
;
Models, Biological
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Mutation
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Neocortex
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cytology
;
growth & development
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metabolism
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Nerve Tissue Proteins
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deficiency
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genetics
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Neurogenesis
;
genetics
;
Neurons
;
cytology
;
metabolism
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Organoids
;
cytology
;
metabolism
;
PAX6 Transcription Factor
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genetics
;
metabolism
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Patch-Clamp Techniques
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SOXB1 Transcription Factors
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genetics
;
metabolism
;
Zonula Occludens-1 Protein
;
genetics
;
metabolism
4.Curcumin induces differentiation of embryonic stem cells through possible modulation of nitric oxide-cyclic GMP pathway.
Kalpana MUJOO ; Lubov E NIKONOFF ; Vladislav G SHARIN ; Nathan S BRYAN ; Alexander Y KOTS ; Ferid MURAD
Protein & Cell 2012;3(7):535-544
Curcumin, an active ingredient of dietary spice used in curry, has been shown to exhibit anti-oxidant, anti-inflammatory and anti-proliferative properties. Using EB directed differentiation protocol of H-9 human embryonic stem (ES) cells; we evaluated the effect of curcumin (0-20 μmol/L) in enhancing such differentiation. Our results using real time PCR, western blotting and immunostaining demonstrated that curcumin significantly increased the gene expression and protein levels of cardiac specific transcription factor NKx2.5, cardiac troponin I, myosin heavy chain, and endothelial nitric oxide synthase during ES cell differentiation. Furthermore, an NO donor enhanced the curcumin-mediated induction of NKx2.5 and other cardiac specific proteins. Incubation of cells with curcumin led to a dose dependent increase in intracellular nitrite to the same extent as giving an authentic NO donor. Functional assay for second messenger(s) cyclic AMP (cAMP) and cyclic GMP (cGMP) revealed that continuous presence of curcumin in differentiated cells induced a decrease in the baseline levels of cAMP but it significantly elevated baseline contents of cGMP. Curcumin addition to a cell free assay significantly suppressed cAMP and cGMP degradation in the extracts while long term treatment of intact cells with curcumin increased the rates of cAMP and cGMP degradation suggesting that this might be due to direct suppression of some cyclic nucleotide-degrading enzyme (phosphodiesterase) by curcumin. These studies demonstrate that polyphenol curcumin may be involved in differentiation of ES cells partly due to manipulation of nitric oxide signaling.
Animals
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Antioxidants
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pharmacology
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Cell Differentiation
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drug effects
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Cells, Cultured
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Curcumin
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pharmacology
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Cyclic GMP
;
metabolism
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Embryoid Bodies
;
drug effects
;
metabolism
;
physiology
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Enzyme Activators
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pharmacology
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Gene Expression
;
drug effects
;
Guanylate Cyclase
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genetics
;
metabolism
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Homeobox Protein Nkx-2.5
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Homeodomain Proteins
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genetics
;
metabolism
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Humans
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Mice
;
Myosin Heavy Chains
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genetics
;
metabolism
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Nitric Oxide
;
metabolism
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Nitric Oxide Donors
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pharmacology
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Nitric Oxide Synthase Type III
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genetics
;
metabolism
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Nitroso Compounds
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pharmacology
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Pyrazoles
;
pharmacology
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Pyridines
;
pharmacology
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Second Messenger Systems
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Transcription Factors
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genetics
;
metabolism
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Troponin
;
genetics
;
metabolism
;
Tumor Suppressor Protein p53
;
metabolism