■人诱导多能干细胞(iPSCs),它们能够从患者身上产生人类神经细胞(星形胶质细胞和神经元),对了解精神分裂症和双相情感障碍等主要神经精神疾病的病理生理学有很大的希望,其中包括大脑发育的改变。的确,iPSCs的体外神经分化,在概括体内神经发育的某些主要阶段的同时,使得获得活的人类神经元网络成为可能。提出的培养模型在这个框架内特别有吸引力,因为它涉及iPSC衍生的神经细胞,更具体地分化为不同类型的皮质神经元(特别是谷氨酸能和GABA能)和星形胶质细胞。然而,这些体外神经元网络,它们的分化程度可能是异质的,要达到适当的成熟水平仍然具有挑战性。因此,有必要开发能够分析大量细胞的工具来评估这种成熟过程。钙(Ca2+)成像,已经得到了广泛的发展,无疑提供了一个非常好的方法,特别是在其版本中使用基因编码的钙指标。然而,在这些iPSC衍生的神经细胞培养的背景下,缺乏能够很好地表征神经分化过程中神经元成熟进化的Ca2+成像方法的研究。
■在这项研究中,我们提出了一种稳健而可靠的方法,用于在这种人类神经培养物中的神经分化过程的两个不同时间点特异性测量神经元活动。为此,我们开发了一种特定的Ca2信号分析程序,并测试了一系列不同的AAV血清型,以在神经元特异性人突触素1(hSyn)启动子的控制下获得GCaMP6f的表达水平。
■已发现反血清型在驱动GCaMP6f的表达方面是最有效的,并且与我们的人iPSC衍生的神经培养物中的多时间点神经元Ca2+成像相容。在hSyn启动子下携带GCaMP6f的AAV2/retro(AAV2/retro-hSyn-GCaMP6f)是我们已经鉴定的有效载体。建立方法,在神经分化过程中的两个时间点使用hSyn和CAG启动子进行钙测量,已知后者提供跨各种细胞类型的高瞬时基因表达。
■我们的结果强调,这种涉及AAV2/retro-hSyn-GCaMP6f的方法适用于在多个时间点特异性测量神经元钙活性,并且与我们混合的人类神经培养物中的神经分化过程兼容。
UNASSIGNED: Human induced pluripotent stem cells (iPSCs), with their ability to generate human neural cells (astrocytes and neurons) from patients, hold great promise for understanding the pathophysiology of major neuropsychiatric diseases such as schizophrenia and bipolar disorders, which includes alterations in cerebral development. Indeed, the in vitro
neurodifferentiation of iPSCs, while recapitulating certain major stages of neurodevelopment in vivo, makes it possible to obtain networks of living human neurons. The culture model presented is particularly attractive within this framework since it involves iPSC-derived neural cells, which more specifically differentiate into cortical neurons of diverse types (in particular glutamatergic and GABAergic) and astrocytes. However, these in vitro neuronal networks, which may be heterogeneous in their degree of differentiation, remain challenging to bring to an appropriate level of maturation. It is therefore necessary to develop tools capable of analyzing a large number of cells to assess this maturation process. Calcium (Ca2+) imaging, which has been extensively developed, undoubtedly offers an incredibly good approach, particularly in its versions using genetically encoded calcium indicators. However, in the context of these iPSC-derived neural cell cultures, there is a lack of studies that propose Ca2+ imaging methods that can finely characterize the evolution of neuronal maturation during the
neurodifferentiation process.
UNASSIGNED: In this study, we propose a robust and reliable method for specifically measuring neuronal activity at two different time points of the
neurodifferentiation process in such human neural cultures. To this end, we have developed a specific Ca2+ signal analysis procedure and tested a series of different AAV serotypes to obtain expression levels of GCaMP6f under the control of the neuron-specific human synapsin1 (hSyn) promoter.
UNASSIGNED: The retro serotype has been found to be the most efficient in driving the expression of the GCaMP6f and is compatible with multi-time point neuronal Ca2+ imaging in our human iPSC-derived neural cultures. An AAV2/retro carrying GCaMP6f under the hSyn promoter (AAV2/retro-hSyn-GCaMP6f) is an efficient vector that we have identified. To establish the method, calcium measurements were carried out at two time points in the
neurodifferentiation process with both hSyn and CAG promoters, the latter being known to provide high transient gene expression across various cell types.
UNASSIGNED: Our results stress that this methodology involving AAV2/retro-hSyn-GCaMP6f is suitable for specifically measuring neuronal calcium activities over multiple time points and is compatible with the
neurodifferentiation process in our mixed human neural cultures.