高效诱导人胚胎干细胞分化为表达生殖功能调控相关基因的下丘脑神经细胞
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摘要
目的优化体外诱导人胚胎干细胞向类下丘脑弓状核神经细胞分化方案、提高分化效率;明确分化神经细胞中是否表达生殖功能调控相关基因。方法利用接受辅助生殖技术治疗患者捐赠的冷冻胚胎建立人胚胎干细胞系CCRM14;饲养层上培养CCRM14,经纯化后消化为单细胞、诱导其向下丘脑神经细胞分化。分化策略:神经分化早期抑制转化生长因子β和骨形态发生蛋白信号通路;接着激活sonic hedgehog信号通路,促使下丘脑神经前体细胞生成;随后抑制Notch信号通路,添加脑源性神经营养因子诱导类下丘脑弓状核神经细胞的分化与成熟。分化不同时期采样,利用RT-qPCR、细胞免疫荧光、流式细胞术方法鉴定分化细胞。结果依据本优化分化方案,CCRM14分化为下丘脑神经前体细胞的效率达95%以上,分化的细胞表达下丘脑弓状核神经细胞特异性标志物POMC和NPY/AGRP,同时表达参与生殖功能调控基因KISS1和AR。结论该类分化细胞有作为细胞模型用于研究下丘脑弓状核在生殖相关疾病发生过程中调控机制的潜力。
Objective: To optimize the protocol for differentiation of human embryonic stem cells into hypothalamic arcuate-like neurons in vitro,and determine whether or not the genes related to regulation of reproductive function were expressed in differentiated hypothalamic arcuate neurons.Methods:Human embryonic stem cell line CCRM14 was generated from frozen embryos donated by patients with assisted reproductive technology.CCRM14 cells were cultured on a layer of feeder cells,dissociated to single cells after purified,and then induced into hypothalamic neurons.The first step of inducing differentiation of human embryonic stem cells into neuronal was inhibition of transforming growth factor-β(TGF-β)and bone morphogenetic protein(BMP)signaling pathways.The next step was activation of sonic hedgehog(SHH)signaling to promote generation of hypothalamic neuronal progenitors.Finally,Notch signaling pathway was inhibited,and brain-derived neurotrophic factor(BDNF)was added to induce differentiation and maturation of hypothalamic arcuate-like neurons.Cell samples were collected at different stages of differentiation.RT-qPCR and cellular immunofluorescence and flow cytometry were used to identify differentiated cells.Results: According to the optimized differentiation protocol,the efficiency of differentiation of hypothalamic neural progenitors from CCRM14 was above 95%.The differentiated hypothalamic arcuatelike neurons expressed POMC and NPY/AGRP which are the specific markers of hypothalamic arcuate neurons,and expressed KISS1 and AR which are involved in the regulation of reproductive function.Conclusions:These differentiated neurons have potential to be a cellular model for further studies on regulatory mechanism of hypothalamic arcuate neurons in the process of reproductive diseases.
Objective: To optimize the protocol for differentiation of human embryonic stem cells into hypothalamic arcuate-like neurons in vitro,and determine whether or not the genes related to regulation of reproductive function were expressed in differentiated hypothalamic arcuate neurons.Methods:Human embryonic stem cell line CCRM14 was generated from frozen embryos donated by patients with assisted reproductive technology.CCRM14 cells were cultured on a layer of feeder cells,dissociated to single cells after purified,and then induced into hypothalamic neurons.The first step of inducing differentiation of human embryonic stem cells into neuronal was inhibition of transforming growth factor-β(TGF-β)and bone morphogenetic protein(BMP)signaling pathways.The next step was activation of sonic hedgehog(SHH)signaling to promote generation of hypothalamic neuronal progenitors.Finally,Notch signaling pathway was inhibited,and brain-derived neurotrophic factor(BDNF)was added to induce differentiation and maturation of hypothalamic arcuate-like neurons.Cell samples were collected at different stages of differentiation.RT-qPCR and cellular immunofluorescence and flow cytometry were used to identify differentiated cells.Results: According to the optimized differentiation protocol,the efficiency of differentiation of hypothalamic neural progenitors from CCRM14 was above 95%.The differentiated hypothalamic arcuatelike neurons expressed POMC and NPY/AGRP which are the specific markers of hypothalamic arcuate neurons,and expressed KISS1 and AR which are involved in the regulation of reproductive function.Conclusions:These differentiated neurons have potential to be a cellular model for further studies on regulatory mechanism of hypothalamic arcuate neurons in the process of reproductive diseases.
引文
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[27]Reinhardt P,Schmid B,Burbulla LF,et al.Genetic correction of a LRRK2 mutation in human iPSCs links parkinsonian neurodegeneration to ERK-dependent changes in gene expression[J].Cell Stem Cell,2013,12:354-367.
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[2]Gao Y,Sun T.Molecular regulation of hypothalamic development and physiological functions[J].Mol Neurobiol,2016,53:4275-4285.
[3]Barsh GS,Schwartz MW.Genetic approaches to studying energy balance:perception and integration[J].Nat Rev Genet,2002,3:589-600.
[4]Sohn JW.Network of hypothalamic neurons that control appetite[J].BMB Rep,2015,48:229-233.
[5]Qiu J,Nestor CC,Zhang C,et al.High-frequency stimulationinduced peptide release synchronizes arcuate kisspeptin neurons and excites GnRH neurons[J].Elife,2016,5:e16246.
[6]CJL H,Kavanagh GS,Smith JT.The role of kisspeptin neurons in reproduction and metabolism[J].J Endocrinol,2018,238:R173-R183.
[7]李江源.奇士肽神经内分泌网络与生殖功能调控[J].生殖医学杂志,2017,26:99-105.
[8]De Bond JA,Smith JT.Kisspeptin and energy balance in reproduction[J].Reproduction,2014,147:R53-63.
[9]Wahab F,Atika B,Ullah F,et al.Metabolic impact on the hypothalamic Kisspeptin-Kiss1rsignaling pathway[J].Front Endocrinol(Lausanne),2018,9:123.
[10]Tolson KP,Garcia C,Yen S,et al.Impaired kisspeptin signaling decreases metabolism and promotes glucose intolerance and obesity[J].J Clin Invest,2014,124:3075-3079.
[11]Castellano JM,Navarro VM,Fernández-Fernández R,et al.Changes in hypothalamic KiSS-1system and restoration of pubertal activation of the reproductive axis by kisspeptin in undernutrition[J].Endocrinology,2005,146:3917-3925.
[12]Iwasa T,Matsuzaki T,Yano K,et al.Effects of Low Energy Availability on Reproductive Functions and Their Underlying Neuroendocrine Mechanisms[J].J Clin Med,2018,7:E166.
[13]张琴静,覃莲菊,崔毓桂,等.干细胞向生殖细胞分化的研究进展[J].生殖医学杂志,2016,25:478-481.
[14]Thomson JA,Itskovitz-Eldor J,Shapiro SS,et al.Embryonic stem cell lines derived from human blastocysts[J].Science,1998,282:1145-1147.
[15]Takahashi K,Yamanaka S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J].Cell,2006,126:663-676.
[16]Wataya T,Ando S,Muguruma K,et al.Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation[J].Proc Natl Acad Sci USA,2008,105:11796-11801.
[17]Merkle FT,Maroof A,Wataya T,et al.Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells[J].Development,2015,142:633-643.
[18]Wang L,Meece K,Williams DJ,et al.Differentiation of hypothalamic-like neurons from human pluripotent stem cells[J].J Clin Invest,2015,125:796-808.
[19]Wang L,Egli D,Leibel RL.Efficient Generation of Hypothalamic Neurons from Human Pluripotent Stem Cells[J].Curr Protoc Hum Genet,2016,90:21.5.1-21.5.14.
[20]Rajamani U,Gross AR,Hjelm BE,et al.Super-Obese PatientDerived iPSC Hypothalamic Neurons Exhibit Obesogenic Signatures and Hormone Responses[J].Cell Stem Cell,2018,22:698-712.
[21]白爱红,孙莹璞,王芳,等.利用冷冻胚胎建立人胚胎干细胞系[J].生殖医学杂志,2010,19:506-510.
[22]Kirwan P,Jura M,Merkle FT.Generation and Characterization of Functional Human Hypothalamic Neurons[J].Curr Protoc Neurosci,2017,81:3.33.1-3.33.24.
[23]邱佳慧,谭季春.胚胎干细胞与遗传病模型的构建[J].中国组织工程研究,2018,22:2769-2774.
[24]Boissart C,Poulet A,Georges P,et al.Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening[J].Transl Psychiatry,2013,3:e294.
[25]Yamada-Goto N,Ochi Y,Katsuura G,et al.Neuronal cells derived from human induced pluripotent stem cells as a functional tool of melanocortin system[J].Neuropeptides,2017,65:10-20.
[26]Fuller HR,Mandefro B,Shirran SL,et al.Spinal Muscular Atrophy Patient iPSC-Derived Motor Neurons Have Reduced Expression of Proteins Important in Neuronal Development[J].Front Cell Neurosci,2015,9:506.
[27]Reinhardt P,Schmid B,Burbulla LF,et al.Genetic correction of a LRRK2 mutation in human iPSCs links parkinsonian neurodegeneration to ERK-dependent changes in gene expression[J].Cell Stem Cell,2013,12:354-367.
[28]Gu M,Mordwinkin NM,Kooreman NG,et al.Pravastatin reverses obesity-induced dysfunction of induced pluripotent stem cell-derived endothelial cells via a nitric oxide-dependent mechanism[J].Eur Heart J,2015,36:806-816.