人胚胎干细胞源多巴胺能神经元的功能性分化
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摘要
背景:干细胞源多巴胺能神经元作为帕金森病替代疗法的细胞来源,其体外分化方案被不断的优化和改良,后续的鉴定手段和检测指标也随之不断完善。目的:通过观察人胚胎干细胞分化为多巴胺能神经元的形态发育过程,检测其电生理特性,以了解在目前分化方案下人胚胎干细胞能否发育为形态成熟、功能成熟的多巴胺能神经元。方法:通过单层贴壁法,采用SMAD通道双抑制剂分化方案,定向诱导人胚胎干细胞分化为多巴胺能神经元,通过光镜、电镜和细胞免疫荧光等检测手段进行形态学和免疫化学鉴定,并应用膜片钳技术检测多巴胺能神经元的电生理特性。参照体内多巴胺能神经元的电生理标准,对体外分化的多巴胺能神经元进行功能评价。结果与结论:(1)采用SMAD通道双抑制剂分化方案,成功诱导人胚胎干细胞定向分化为形态学上发育成熟的多巴胺能神经元;(2)膜片钳检测结果显示多巴胺能神经元具有成熟的电生理功能,且其电生理特性符合体内多巴胺能神经元的评价标准;(3)上述分化方案可以将人胚胎干细胞定向分化为成熟的、有功能的多巴胺能神经元。
BACKGROUND: The in vitro differentiation methods of stem cell-derived dopaminergic neurons that serve as a cell source for the replacement therapy of Parkinson's disease are continuously optimized and improved, as well as the subsequent identification methods and testing indicators. OBJECTIVE: To observe the morphological development and electrophysiological characteristics of dopaminergic neurons differentiated from human embryonic stem cells so as to identify whether these differentiated cells have mature morphology and function under the current differentiation program. METHODS: Monolayer adherent method combined with dual-SMAD signaling inhibition was used to induce the directional differentiation of human embryonic stem cells into dopaminergic neurons. Then the cells were identified by light microscopy, electron microscopy and immunofluorescence, and the electrophysiological properties of dopaminergic neurons were detected by patch clamp electrophysiological technique. Herein, we evaluated the electrophysiological functions of dopaminergic neurons differentiated in vitro, with reference to the evaluation standard of dopaminergic neuron in vivo. RESULTS AND CONCLUSION: In this study, we successfully obtained dopaminergic neurons with mature morphology and functions differentiated from human embryonic stem cells in vitro. Findings from the subsequent electrophysiological test confirmed that dopaminergic neurons we acquired had electrophysiological properties in accordance with the evaluation standards of dopaminergic neurons in vivo. To conclude, the monolayer adherent method combined with dual-SMAD signaling inhibition can successfully induce the directional differentiation of human embryonic stem cells into dopaminergic neurons with mature morphology and functions.
BACKGROUND: The in vitro differentiation methods of stem cell-derived dopaminergic neurons that serve as a cell source for the replacement therapy of Parkinson's disease are continuously optimized and improved, as well as the subsequent identification methods and testing indicators. OBJECTIVE: To observe the morphological development and electrophysiological characteristics of dopaminergic neurons differentiated from human embryonic stem cells so as to identify whether these differentiated cells have mature morphology and function under the current differentiation program. METHODS: Monolayer adherent method combined with dual-SMAD signaling inhibition was used to induce the directional differentiation of human embryonic stem cells into dopaminergic neurons. Then the cells were identified by light microscopy, electron microscopy and immunofluorescence, and the electrophysiological properties of dopaminergic neurons were detected by patch clamp electrophysiological technique. Herein, we evaluated the electrophysiological functions of dopaminergic neurons differentiated in vitro, with reference to the evaluation standard of dopaminergic neuron in vivo. RESULTS AND CONCLUSION: In this study, we successfully obtained dopaminergic neurons with mature morphology and functions differentiated from human embryonic stem cells in vitro. Findings from the subsequent electrophysiological test confirmed that dopaminergic neurons we acquired had electrophysiological properties in accordance with the evaluation standards of dopaminergic neurons in vivo. To conclude, the monolayer adherent method combined with dual-SMAD signaling inhibition can successfully induce the directional differentiation of human embryonic stem cells into dopaminergic neurons with mature morphology and functions.
引文
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[28]Yan Y,Yang D,Zarnowska ED,et al.Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells.Stem Cells.2005;23(6):781-790.
[29]Schulz TC,Noggle SA,Palmarini GM,et al.Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.Stem Cells.2004;22(7):1218-1238.
[30]Park S,Lee KS,Lee YJ,et al.Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors.Neurosci Lett.2004;359(1-2):99-103.
[31]Cho MS,Lee YE,Kim JY,et al.Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells.Proc Natl Acad Sci U S A.2008;105(9):3392-3397.
[32]Yang F,Liu Y,Tu J,et al.Activated astrocytes enhance the dopaminergic differentiation of stem cells and promote brain repair through b FGF.Nat Commun.2014;5:5627.
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[36]Lahti L,Peltopuro P,Piepponen TP,et al.Cell-autonomous FGF signaling regulates anteroposterior patterning and neuronal differentiation in the mesodiencephalic dopaminergic progenitor domain.Development.2012;139(5):894-905.
[37]Cooper O,Hargus G,Deleidi M,et al.Differentiation of human ES and Parkinson's disease i PS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH,FGF8a and specific regionalization by retinoic acid.Mol Cell Neurosci.2010;45(3):258-266.
[38]Chambers SM,Fasano CA,Papapetrou EP,et al.Highly efficient neural conversion of human ES and i PS cells by dual inhibition of SMAD signaling.Nat Biotechnol.2009;27(3):275-280.
[39]Morizane A,Doi D,Kikuchi T,et al.Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells.J Neurosci Res.2011;89(2):117-126.
[40]Cai J,Schleidt S,Pelta-Heller J,et al.BMP and TGF-βpathway mediators are critical upstream regulators of Wnt signaling during midbrain dopamine differentiation in human pluripotent stem cells.Dev Biol.2013;376(1):62-73.
[41]He XB,Kim M,Kim SY,et al.Vitamin C facilitates dopamine neuron differentiation in fetal midbrain through TET1-and JMJD3-dependent epigenetic control manner.Stem Cells.2015;33(4):1320-1332.
[42]Ning H,Huang YC,Banie L,et al.Micro RNA regulation of neuron-like differentiation of adipose tissue-derived stem cells.Differentiation.2009;78(5):253-259.
[43]Coyne L,Shan M,Przyborski SA,et al.Neuropharmacological properties of neurons derived from human stem cells.Neurochem Int.2011;59(3):404-412.
[44]Westerlund U,Moe MC,Varghese M,et al.Stem cells from the adult human brain develop into functional neurons in culture.Exp Cell Res.2003;289(2):378-383.
[45]Xi J,Liu Y,Liu H,et al.Specification of midbrain dopamine neurons from primate pluripotent stem cells.Stem Cells.2012;30(8):1655-1663.
[46]Stanslowsky N,Haase A,Martin U,et al.Functional differentiation of midbrain neurons from human cord blood-derived induced pluripotent stem cells.Stem Cell Res Ther.2014;5(2):35.
[47]Sagal J,Zhan X,Xu J,et al.Proneural transcription factor Atoh1drives highly efficient differentiation of human pluripotent stem cells into dopaminergic neurons.Stem Cells Transl Med.2014;3(8):888-898.
[48]Donato R,Miljan EA,Hines SJ,et al.Differential development of neuronal physiological responsiveness in two human neural stem cell lines.BMC Neurosci.2007;8:36.
[49]Ding S,Wei W,Zhou FM.Molecular and functional differences in voltage-activated sodium currents between GABA projection neurons and dopamine neurons in the substantia nigra.J Neurophysiol.2011;106(6):3019-3034.
[50]Seutin V,Engel D.Differences in Na+conductance density and Na+channel functional properties between dopamine and GABA neurons of the rat substantia nigra.J Neurophysiol.2010;103(6):3099-3114.
[51]Vazin T,Becker KG,Chen J,et al.A novel combination of factors,termed SPIE,which promotes dopaminergic neuron differentiation from human embryonic stem cells.PLo S One.2009;4(8):e6606.
[52]Schulz TC,Noggle SA,Palmarini GM,et al.Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.Stem Cells.2004;22(7):1218-1238.
[53]Hermann A,Maisel M,Wegner F,et al.Multipotent neural stem cells from the adult tegmentum with dopaminergic potential develop essential properties of functional neurons.Stem Cells.2006;24(4):949-964.
[2]Damier P,Hirsch EC,Agid Y,et al.The substantia nigra of the human brain.II.Patterns of loss of dopamine-containing neurons in Parkinson's disease.Brain.1999;122(Pt 8):1437-1448.
[3]Zhang Q,Chen W,Tan S,et al.Stem Cells for Modeling and Therapy of Parkinson's Disease.Hum Gene Ther.2017;28(1):85-98.
[4]Jenner P.Dopamine agonists,receptor selectivity and dyskinesia induction in Parkinson's disease.Curr Opin Neurol.2003;16 Suppl 1:S3-7.
[5]Huot P,Johnston TH,Koprich JB,et al.The pharmacology of L-DOPA-induced dyskinesia in Parkinson's disease.Pharmacol Rev.2013;65(1):171-222.
[6]Barker RA,Drouin-Ouellet J,Parmar M.Cell-based therapies for Parkinson disease—past insights and future potential.Nat Rev Neurol.2015;11(9):492-503.
[7]Ziavra D,Makri G,Giompres P,et al.Neural stem cells transplanted in a mouse model of Parkinson's disease differentiate to neuronal phenotypes and reduce rotational deficit.CNS Neurol Disord Drug Targets.2012;11(7):829-835.
[8]Muraoka K,Shingo T,Yasuhara T,et al.Comparison of the therapeutic potential of adult and embryonic neural precursor cells in a rat model of Parkinson disease.J Neurosurg.2008;108(1):149-159.
[9]He XB,Kim M,Kim SY,et al.Vitamin C facilitates dopamine neuron differentiation in fetal midbrain through TET1-and JMJD3-dependent epigenetic control manner.Stem Cells.2015;33(4):1320-1332.
[10]Kim JH,Auerbach JM,Rodríguez-Gómez JA,et al.Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease.Nature.2002;418(6893):50-56.
[11]Dahlstroem A,Fuxe K.Evidence for the existence of monoamine-containing neurons in the central nervous system.i.demonstration of monoamines in the cell bodies of brain stem neurons.Acta Physiol Scand Suppl.1964:SUPPL 232:1-55.
[12]Lees AJ,Hardy J,Revesz T.Parkinson's disease.Lancet.2009;373(9680):2055-2066.
[13]Tzschentke TM,Schmidt WJ.Functional relationship among medial prefrontal cortex,nucleus accumbens,and ventral tegmental area in locomotion and reward.Crit Rev Neurobiol.2000;14(2):131-142.
[14]Grace AA,Bunney BS.The control of firing pattern in nigral dopamine neurons:burst firing.J Neurosci.1984;4(11):2877-2890.
[15]Grace AA,Bunney BS.The control of firing pattern in nigral dopamine neurons:single spike firing.J Neurosci.1984;4(11):2866-2876.
[16]Grace AA,Onn SP.Morphology and electrophysiological properties of immunocytochemically identified rat dopamine neurons recorded in vitro.J Neurosci.1989;9(10):3463-3481.
[17]Hainsworth AH,R?per J,Kapoor R,et al.Identification and electrophysiology of isolated pars compacta neurons from guinea-pig substantia nigra.Neuroscience.1991;43(1):81-93.
[18]Ungless MA,Grace AA.Are you or aren't you?Challenges associated with physiologically identifying dopamine neurons.Trends Neurosci.2012;35(7):422-430.
[19]Good CH,Hoffman AF,Hoffer BJ,et al.Impaired nigrostriatal function precedes behavioral deficits in a genetic mitochondrial model of Parkinson's disease.FASEB J.2011;25(4):1333-1344.
[20]Branch SY,Chen C,Sharma R,et al.Dopaminergic Neurons Exhibit an Age-Dependent Decline in Electrophysiological Parameters in the Mito Park Mouse Model of Parkinson's Disease.J Neurosci.2016;36(14):4026-4037.
[21]Oh JE,Bae GU,Yang YJ,et al.Cdo promotes neuronal differentiation via activation of the p38 mitogen-activated protein kinase pathway.FASEB J.2009;23(7):2088-2099.
[22]Lee HS,Bae EJ,Yi SH,et al.Foxa2 and Nurr1 synergistically yield A9 nigral dopamine neurons exhibiting improved differentiation,function,and cell survival.Stem Cells.2010;28(3):501-512.
[23]Takaesu G,Kang JS,Bae GU,et al.Activation of p38alpha/beta MAPK in myogenesis via binding of the scaffold protein JLP to the cell surface protein Cdo.J Cell Biol.2006;175(3):383-388.
[24]Perrier AL,Tabar V,Barberi T,et al.Derivation of midbrain dopamine neurons from human embryonic stem cells.Proc Natl Acad Sci U S A.2004;101(34):12543-12548.
[25]Park CH,Minn YK,Lee JY,et al.In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons.J Neurochem.2005;92(5):1265-1276.
[26]Buytaert-Hoefen KA,Alvarez E,Freed CR.Generation of tyrosine hydroxylase positive neurons from human embryonic stem cells after coculture with cellular substrates and exposure to GDNF.Stem Cells.2004;22(5):669-674.
[27]Zeng X,Cai J,Chen J,et al.Dopaminergic differentiation of human embryonic stem cells.Stem Cells.2004;22(6):925-940.
[28]Yan Y,Yang D,Zarnowska ED,et al.Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells.Stem Cells.2005;23(6):781-790.
[29]Schulz TC,Noggle SA,Palmarini GM,et al.Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.Stem Cells.2004;22(7):1218-1238.
[30]Park S,Lee KS,Lee YJ,et al.Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors.Neurosci Lett.2004;359(1-2):99-103.
[31]Cho MS,Lee YE,Kim JY,et al.Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells.Proc Natl Acad Sci U S A.2008;105(9):3392-3397.
[32]Yang F,Liu Y,Tu J,et al.Activated astrocytes enhance the dopaminergic differentiation of stem cells and promote brain repair through b FGF.Nat Commun.2014;5:5627.
[33]Bayly RD,Brown CY,Agarwala S.A novel role for FOXA2 and SHH in organizing midbrain signaling centers.Dev Biol.2012;369(1):32-42.
[34]Andersson E,Tryggvason U,Deng Q,et al.Identification of intrinsic determinants of midbrain dopamine neurons.Cell.2006;124(2):393-405.
[35]L'Episcopo F,Tirolo C,Testa N,et al.Wnt/β-catenin signaling is required to rescue midbrain dopaminergic progenitors and promote neurorepair in ageing mouse model of Parkinson's disease.Stem Cells.2014;32(8):2147-2163.
[36]Lahti L,Peltopuro P,Piepponen TP,et al.Cell-autonomous FGF signaling regulates anteroposterior patterning and neuronal differentiation in the mesodiencephalic dopaminergic progenitor domain.Development.2012;139(5):894-905.
[37]Cooper O,Hargus G,Deleidi M,et al.Differentiation of human ES and Parkinson's disease i PS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH,FGF8a and specific regionalization by retinoic acid.Mol Cell Neurosci.2010;45(3):258-266.
[38]Chambers SM,Fasano CA,Papapetrou EP,et al.Highly efficient neural conversion of human ES and i PS cells by dual inhibition of SMAD signaling.Nat Biotechnol.2009;27(3):275-280.
[39]Morizane A,Doi D,Kikuchi T,et al.Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells.J Neurosci Res.2011;89(2):117-126.
[40]Cai J,Schleidt S,Pelta-Heller J,et al.BMP and TGF-βpathway mediators are critical upstream regulators of Wnt signaling during midbrain dopamine differentiation in human pluripotent stem cells.Dev Biol.2013;376(1):62-73.
[41]He XB,Kim M,Kim SY,et al.Vitamin C facilitates dopamine neuron differentiation in fetal midbrain through TET1-and JMJD3-dependent epigenetic control manner.Stem Cells.2015;33(4):1320-1332.
[42]Ning H,Huang YC,Banie L,et al.Micro RNA regulation of neuron-like differentiation of adipose tissue-derived stem cells.Differentiation.2009;78(5):253-259.
[43]Coyne L,Shan M,Przyborski SA,et al.Neuropharmacological properties of neurons derived from human stem cells.Neurochem Int.2011;59(3):404-412.
[44]Westerlund U,Moe MC,Varghese M,et al.Stem cells from the adult human brain develop into functional neurons in culture.Exp Cell Res.2003;289(2):378-383.
[45]Xi J,Liu Y,Liu H,et al.Specification of midbrain dopamine neurons from primate pluripotent stem cells.Stem Cells.2012;30(8):1655-1663.
[46]Stanslowsky N,Haase A,Martin U,et al.Functional differentiation of midbrain neurons from human cord blood-derived induced pluripotent stem cells.Stem Cell Res Ther.2014;5(2):35.
[47]Sagal J,Zhan X,Xu J,et al.Proneural transcription factor Atoh1drives highly efficient differentiation of human pluripotent stem cells into dopaminergic neurons.Stem Cells Transl Med.2014;3(8):888-898.
[48]Donato R,Miljan EA,Hines SJ,et al.Differential development of neuronal physiological responsiveness in two human neural stem cell lines.BMC Neurosci.2007;8:36.
[49]Ding S,Wei W,Zhou FM.Molecular and functional differences in voltage-activated sodium currents between GABA projection neurons and dopamine neurons in the substantia nigra.J Neurophysiol.2011;106(6):3019-3034.
[50]Seutin V,Engel D.Differences in Na+conductance density and Na+channel functional properties between dopamine and GABA neurons of the rat substantia nigra.J Neurophysiol.2010;103(6):3099-3114.
[51]Vazin T,Becker KG,Chen J,et al.A novel combination of factors,termed SPIE,which promotes dopaminergic neuron differentiation from human embryonic stem cells.PLo S One.2009;4(8):e6606.
[52]Schulz TC,Noggle SA,Palmarini GM,et al.Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.Stem Cells.2004;22(7):1218-1238.
[53]Hermann A,Maisel M,Wegner F,et al.Multipotent neural stem cells from the adult tegmentum with dopaminergic potential develop essential properties of functional neurons.Stem Cells.2006;24(4):949-964.