Ganapathy Kavina, Sowmithra Sowmithra, Bhonde Ramesh, Datta Indrani
Cells Tissues Organs. 2015;201(6):445-463. doi: 10.1159/000446424. Epub 2016 Jul 16.
The neuron-glia ratio is of prime importance for maintaining the physiological homeostasis of neuronal and glial cells, and especially crucial for dopaminergic neurons because a reduction in glial density has been reported in postmortem reports of brains affected by Parkinson's disease. We thus aimed at developing an in vitro midbrain culture which would replicate a similar neuron-glia ratio to that in in vivo adult midbrain while containing a similar number of dopaminergic neurons. A sequential culture technique was adopted to achieve this. Neural progenitors (NPs) were generated by the hanging-drop method and propagated as 3D neurospheres followed by the derivation of outgrowth from these neurospheres on a chosen extracellular matrix. The highest proliferation was observed in neurospheres from day in vitro (DIV) 5 through MTT and FACS analysis of Ki67 expression. FACS analysis using annexin/propidium iodide showed an increase in the apoptotic population from DIV 8. DIV 5 neurospheres were therefore selected for deriving the differentiated outgrowth of midbrain on a poly-L-lysine-coated surface. Quantitative RT-PCR showed comparable gene expressions of the mature neuronal marker β-tubulin III, glial marker GFAP and dopaminergic marker tyrosine hydroxylase (TH) as compared to in vivo adult rat midbrain. The FACS analysis showed a similar neuron-glia ratio obtained by the sequential culture in comparison to adult rat midbrain. The yield of β-tubulin III and TH was distinctly higher in the sequential culture in comparison to 2D culture, which showed a higher yield of GFAP immunopositive cells. Functional characterization indicated that both the constitutive and inducible (KCl and ATP) release of dopamine was distinctly higher in the sequential culture than the 2D culture. Thus, the sequential culture technique succeeded in the initial enrichment of NPs in 3D neurospheres, which in turn resulted in an optimal attainment of the neuron-glia ratio on outgrowth culture from these neurospheres.
神经元与神经胶质细胞的比例对于维持神经元和神经胶质细胞的生理稳态至关重要，对于多巴胺能神经元尤为关键，因为在帕金森病患者的尸检报告中已报道神经胶质密度降低。因此，我们旨在开发一种体外中脑培养方法，该方法能复制与体内成年中脑相似的神经元与神经胶质细胞比例，同时包含相似数量的多巴胺能神经元。为此采用了一种序贯培养技术。通过悬滴法生成神经祖细胞（NP），并将其作为三维神经球进行增殖，随后从这些神经球在选定的细胞外基质上诱导长出突起。通过MTT以及对Ki67表达的流式细胞术分析，观察到从体外培养第5天起神经球的增殖最为旺盛。使用膜联蛋白/碘化丙啶的流式细胞术分析显示，从第8天起凋亡细胞群体增加。因此选择第5天的神经球在聚-L-赖氨酸包被的表面诱导中脑分化长出突起。定量逆转录聚合酶链反应显示，与体内成年大鼠中脑相比，成熟神经元标志物β-微管蛋白III、神经胶质标志物胶质纤维酸性蛋白（GFAP）和多巴胺能标志物酪氨酸羟化酶（TH）的基因表达相当。流式细胞术分析显示，序贯培养获得的神经元与神经胶质细胞比例与成年大鼠中脑相似。与二维培养相比，序贯培养中β-微管蛋白III和TH的产量明显更高，二维培养中GFAP免疫阳性细胞的产量更高。功能特性表明，序贯培养中多巴胺的组成型和诱导型（氯化钾和三磷酸腺苷）释放明显高于二维培养。因此，序贯培养技术成功地在三维神经球中初步富集了NP，进而在从这些神经球长出的培养物中实现了神经元与神经胶质细胞比例的最佳状态。
