目的 探讨GSK3α和GSK3β在胚胎干细胞体外神经分化中的作用。 方法 利用CRISPR/Cas9技术构建Gsk3a或Gsk3b敲除的胚胎干细胞，在体外对其进行神经诱导分化，并通过转录组测序（RNA-seq）分析胚胎干细胞的基因表达差异。 结果 Gsk3a-/-或Gsk3b-/-胚胎干细胞仍然具有多能性，能够分化成神经干细胞。然而，Gsk3b-/-无法形成神经花环，其神经干细胞也不能进一步分化成神经元和胶质细胞。检测三胚层标志物的mRNA表达发现，Gsk3a-/-神经外胚层标志物的表达略高于野生（WT）组，而Gsk3b-/-内胚层标志物的表达远高于WT组和Gsk3a-/-（P<0.001），神经外胚层标志物的表达较低。RNA-seq结果显示，涉及JAK-STAT、PI3K-Akt信号通路以及Neurog1、Hes1等基因的变化。 结论 GSK3α抑制神经分化而GSK3β促进神经分化，其机制与JAK-STAT、PI3K-Akt信号通路以及Neurog1、Hes1有关。
Abstract
Objective To investigate the roles of glycogen synthase kinase 3α (GSK3α) and glycogen synthase kinase 3β (GSK3β) in neural differentiation of embryonic stem cells (ESCs) in vitro. Methods Gsk3a or Gsk3b knockout ESCs lines were generated using CRISPR/Cas9 technology. These ESCs lines were then subjected to neural induction in vitro, and the differential gene expression of ESCs was analyzed using RNA sequencing (RNA-seq). Results Gsk3a-/- ESCs or Gsk3b-/- ESCs cells still maintained pluripotency and were capable of differentiating into neural stem cells (NSCs). However, Gsk3b-/- ESCs failed to form neural rosettes, and their NSCs were unable to further differentiate into neurons and glial cells. Analysis of mRNA expression of germ layer markers revealed slightly higher expression of neural ectoderm markers in Gsk3a-/- ESCs compared to the wildtype (WT) group, while the expression of endoderm markers in Gsk3b-/- ESCs was significantly higher than both the WT group and Gsk3a-/- ESCs（P<0.001）, with lower expression of neural ectoderm markers. RNA-seq results indicated changes in genes involved in the JAK-STAT, PI3K-Akt signaling pathway, as well as genes like Neurog1 and Hes1. Conclusions GSK3α inhibits neuronal differentiation while GSK3β promotes neuronal differentiation, and their mechanisms are associated with the JAK-STAT, PI3K-Akt signaling pathways, as well as Neurog1 and Hes1.
关键词
糖原合成激酶3α； /
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糖原合成激酶3β； /
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胚胎干细胞； /
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神经分化
Key words
Glycogen Synthase Kinase 3α; /
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Glycogen Synthase Kinase 3β; /
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Embryonic stem cells /
Neural differentiation
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参考文献
[1] Swain N, Thakur M, Pathak J, et al. SOX2, OCT4 and NANOG: The core embryonic stem cell pluripotency regulators in oral carcinogenesis [J]. J Oral Maxillofac Pathol, 2020, 24(2): 368-373. DOI: 10.4103/jomfp.JOMFP_22_20.
[2] Haddad MS, Wenceslau CV, Pompeia C, et al. Cell-based technologies for Huntington's disease [J]. Dement Neuropsychol, 2016, 10(4): 287-295. DOI: 10.1590/s1980-5764-2016dn1004006.
[3] Sivandzade F, Cucullo L. Regenerative stem cell therapy for neurodegenerative diseases: an overview [J]. Int J Mol Sci, 2021, 22(4). DOI: 10.3390/ijms22042153.
[4] Aly R M. Current state of stem cell-based therapies: an overview [J]. Stem Cell Investig, 2020, 7: 8. DOI: 10.21037/sci-2020-001.
[5] Soutar MPM, Kim W-Y, Williamson R, et al. Evidence that glycogen synthase kinase-3 isoforms have distinct substrate preference in the brain [J]. J Neurochem, 2010, 115(4): 974-983. DOI: 10.1111/j.1471-4159.2010.06988.x.
[6] Ali A, Hoeflich KP, Woodgett JR. Glycogen synthase kinase-3: properties, functions, and regulation [J]. Chem Rev, 2001, 101(8): 2527-2540. DOI: 10.1021/cr000110o.
[7] Alzahrani AS. PI3K/Akt/mTOR inhibitors in cancer: At the bench and bedside [J]. Semin Cancer Biol, 2019, 59: 125-132. DOI: 10.1016/j.semcancer.2019.07.009.
[8] Wang Z, Smith KS, Murphy M, et al. Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy [J]. Nature, 2008, 455(7217): 1205-1209. DOI: 10.1038/nature07284.
[9] Kim W-Y, Snider WD. Functions of GSK-3 Signaling in Development of the Nervous System [J]. Front Mol Neurosci, 2011, 4: 44. DOI: 10.3389/fnmol.2011.00044.
[10] Ying QL, Stavridis M, Griffiths D, et al. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture [J]. Nat Biotechnol, 2003, 21(2): 183-186. DOI: 10.1038/nbt780.
[11] Pankratz MT, Li XJ, Lavaute TM, et al. Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage [J]. Stem Cells (Dayton, Ohio), 2007, 25(6): 1511-1520. DOI: 10.1634/stemcells.2006-0707.
[12] Zhou J, Freeman TA, Ahmad F, et al. GSK-3α is a central regulator of age-related pathologies in mice [J]. J Clin Invest, 2013, 123(4): 1821-1832. DOI: 10.1172/JCI64398.
[13] Kaidanovich-Beilin O, Lipina TV, Takao K, et al. Abnormalities in brain structure and behavior in GSK-3alpha mutant mice [J]. Mol Brain, 2009, 2: 35. DOI: 10.1186/1756-6606-2-35.
[14] Kerkela R, Kockeritz L, Macaulay K, et al. Deletion of GSK-3beta in mice leads to hypertrophic cardiomyopathy secondary to cardiomyoblast hyperproliferation [J]. J Clin Invest, 2008, 118(11): 3609-3618. DOI: 10.1172/JCI36245.
[15] Hedgepeth CM, Conrad LJ, Zhang J, et al. Activation of the Wnt signaling pathway: a molecular mechanism for lithium action [J]. Dev Biol, 1997, 185(1): 82-91. DOI: 10.1006/dbio.1997.8552.
[16] Hooper C, Markevich V, Plattner F, et al. Glycogen synthase kinase-3 inhibition is integral to long-term potentiation [J]. Eur J Neurosci, 2007, 25(1): 81-86. DOI: 10.1111/j.1460-9568.2006.05245.x.
[17] Cao F, Hata R, Zhu P, et al. Conditional deletion of Stat3 promotes neurogenesis and inhibits astrogliogenesis in neural stem cells [J]. Biochem Biophys Res Commun, 2010, 394(3): 843-837. DOI: 10.1016/j.bbrc.2010.03.092.
[18] Tanaka Y, Yasugi T, Nagayama M, et al. JAK/STAT guarantees robust neural stem cell differentiation by shutting off biological noise [J]. Sci Rep, 2018, 8(1): 12484. DOI: 10.1038/s41598-018-30929-1.
[19] Ojeda L, Gao J, Hooten KG, et al. Critical role of PI3K/Akt/GSK3β in motoneuron specification from human neural stem cells in response to FGF2 and EGF [J]. PloS One, 2011, 6(8): e23414. DOI: 10.1371/journal.pone.0023414.
[20] Wilkinson G, Dennis D, Schuurmans C. Proneural genes in neocortical development [J]. Neuroscience, 2013, 253: 256-273. DOI: 10.1016/j.neuroscience.2013.08.029.
[21] Li S, Mattar P, Zinyk D, et al. GSK3 temporally regulates neurogenin 2 proneural activity in the neocortex [J]. J Neurosci, 2012, 32(23): 7791-805. DOI: 10.1523/JNEUROSCI.1309-12.2012.
基金
国家自然科学基金(8207053359)； 广东省自然科学基金（2019A1515011760）
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