Efficient and Cost-Effective Generation of Mature Neurons From Human Induced Pluripotent Stem Cells
Efficient and Cost-Effective Generation of Mature Neurons From Human Induced Pluripotent Stem Cells
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DOI：
10.5966/sctm.2014-0024
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发表时间：
2014-12-01
影响因子：
6
通讯作者：
Magdinier, Frederique
中科院分区：
文献类型：
--
作者：
Badja, Cherif;Maleeva, Galyna;Magdinier, Frederique
多年来，我们研究神经系统疾病病理变化的能力一直受到缺乏相关模型的阻碍，直到Yamanaka小组最近的开创性工作表明，从人体细胞中产生诱导多能干细胞（iPSC）并将这些iPSC的命运重新定向为分化细胞是可行的。特别地，许多兴趣集中在将人iPSC分化成神经元祖细胞和功能性神经元的能力，以与大量病理学（包括精神发育迟滞和行为或退行性综合征）相关。目前的分化方案是耗时的，并且产生有限数量的细胞，阻碍了大规模使用。我们描述了一种无饲养层的方法，依赖于使用化学成分确定的培养基，克服了需要胚状体形成和神经元的前体细胞和终末分化的神经元生产的神经元花结分离。诱导后4天，可检测到神经外胚层谱系标志物的表达。在4至7天之间，神经元前体可以扩增、冷冻和解冻，而不会损失增殖和分化能力或进一步分化。观察到终末分化为人脑中发现的成熟神经元的不同亚型。在诱导后6-35天，细胞表达GABA、甘氨酸和乙酰胆碱的典型电压门控和离子型受体。这种在化学成分确定的培养基中的特异性和有效的单步策略允许在20-40天内产生成熟神经元，具有多种应用，特别是用于模拟人类病理。
For years, our ability to study pathological changes in neurological diseases has been hampered by the lack of relevant models until the recent groundbreaking work from Yamanaka's group showing that it is feasible to generate induced pluripotent stem cells (iPSCs) from human somatic cells and to redirect the fate of these iPSCs into differentiated cells. In particular, much interest has focused on the ability to differentiate human iPSCs into neuronal progenitors and functional neurons for relevance to a large number of pathologies including mental retardation and behavioral or degenerative syndromes. Current differentiation protocols are time-consuming and generate limited amounts of cells, hindering use on a large scale. We describe a feeder-free method relying on the use of a chemically defined medium that overcomes the need for embryoid body formation and neuronal rosette isolation for neuronal precursors and terminally differentiated neuron production. Four days after induction, expression of markers of the neurectoderm lineage is detectable. Between 4 and 7 days, neuronal precursors can be expanded, frozen, and thawed without loss of proliferation and differentiation capacities or further differentiated. Terminal differentiation into the different subtypes of mature neurons found in the human brain were observed. At 6-35 days after induction, cells express typical voltage-gated and ionotrophic receptors for GABA, glycine, and acetylcholine. This specific and efficient single-step strategy in a chemically defined medium allows the production of mature neurons in 20-40 days with multiple applications, especially for modeling human pathologies.