Samata Bumpei, Kikuchi Tetsuhiro, Miyawaki Yoshifumi, Morizane Asuka, Mashimo Tomoji, Nakagawa Masato, Okita Keisuke, Takahashi Jun
Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
J Neurosci Methods. 2015 Mar 30;243:68-77. doi: 10.1016/j.jneumeth.2015.01.027. Epub 2015 Feb 4.
To evaluate the in vivo function of human dopaminergic (DA) neurons, Parkinson's disease (PD) model rats made by the hemi-lateral injection of 6-hydroxydopamine (6-OHDA) are widely used as host animals. In the case of such xeno-transplantation, however, immunosuppression is needed for good survival of the grafted cells.
In order to determine whether human mature neurons can survive in X-linked severe combined immunodeficiency (X-SCID) rats without immunosuppression, we grafted human embryonic stem cell (ESC)-derived DA neurons into the striatum of X-SCID rats. We next treated the X-SCID rats with 6-OHDA and grafted mouse fetal DA neurons or human induced pluripotent stem cell (iPSC)-derived DA neurons to examine whether these rats can be used as PD model rats.
X-SCID rats did not elicit immune responses against human ESC-derived DA neurons and consequently resulted in good survival of the cells without immunosuppression. Furthermore, 6-OHDA-lesioned X-SCID rats exhibited rotational behavior, which was recovered by grafting mouse fetal DA neurons or human iPSC-derived DA neurons.
Immunosuppression by drugs such as Cyclosporine A requires daily injection, which is stressful for rats and moreover may cause renal or hepatic failure. Furthermore, blood levels of the drug may not be stable, which weakens the reliability of the data.
Our results provide a more accessible and reliable method to evaluate the in vivo function of human DA neurons, potentially offering a pre-clinical study for the application of pluripotent stem cells.
为评估人类多巴胺能（DA）神经元的体内功能，通过半侧注射6-羟基多巴胺（6-OHDA）制备的帕金森病（PD）模型大鼠被广泛用作宿主动物。然而，在这种异种移植的情况下，为使移植细胞良好存活需要进行免疫抑制。
为了确定人类成熟神经元在无免疫抑制的X连锁严重联合免疫缺陷（X-SCID）大鼠中能否存活，我们将人胚胎干细胞（ESC）衍生的DA神经元移植到X-SCID大鼠的纹状体中。接下来，我们用6-OHDA处理X-SCID大鼠，并移植小鼠胎儿DA神经元或人诱导多能干细胞（iPSC）衍生的DA神经元，以检查这些大鼠是否可作为PD模型大鼠。
X-SCID大鼠未对人ESC衍生的DA神经元引发免疫反应，因此在无免疫抑制的情况下细胞存活良好。此外，6-OHDA损伤的X-SCID大鼠表现出旋转行为，通过移植小鼠胎儿DA神经元或人iPSC衍生的DA神经元可使其恢复。
使用环孢素A等药物进行免疫抑制需要每日注射，这对大鼠有压力，而且可能导致肾衰竭或肝衰竭。此外，药物的血药浓度可能不稳定，这削弱了数据的可靠性。
我们的结果提供了一种更易操作且可靠的方法来评估人类DA神经元的体内功能，可能为多能干细胞的应用提供临床前研究。
