Albrecht-Kossel-Institute for Neuroregeneration (AKos), University of Rostock, Gehlsheimer Strasse 20, D-18147 Rostock, Germany.
Orphanet J Rare Dis. 2013 Sep 18;8:144. doi: 10.1186/1750-1172-8-144.
Niemann-Pick type C1 disease (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. In this lysosomal storage disorder the intracellular transport and sequestration of several lipids like cholesterol is severely impaired, resulting in an accumulation of lipids in late endosomes and lysosomes. The neurological manifestation of the disease is caused by dysfunction and cell death in the central nervous system. Several animal models were used to analyze the impaired pathways. However, the underlying pathogenic mechanisms are still not completely understood and the genetic variability in humans cannot be reflected in these models. Therefore, a human model using patient-specific induced pluripotent stem cells provides a promising approach.
We reprogrammed human fibroblasts from a NPC1 patient and a healthy control by retroviral transduction with Oct4, Klf4, Sox2 and c-Myc. The obtained human induced pluripotent stem cells (hiPSCs) were characterized by immunocytochemical analyses. Neural progenitor cells were generated and patch clamp recordings were performed for a functional analysis of derived neuronal cells. Filipin stainings and the Amplex Red assay were used to demonstrate and quantify cholesterol accumulation.
The hiPSCs expressed different stem cell markers, e.g. Nanog, Tra-1-81 and SSEA4. Using the embryoid body assay, the cells were differentiated in cells of all three germ layers and induced teratoma in immunodeficient mice, demonstrating their pluripotency. In addition, neural progenitor cells were derived and differentiated into functional neuronal cells. Patch clamp recordings revealed voltage dependent channels, spontaneous action potentials and postsynaptic currents. The accumulation of cholesterol in different tissues is the main hallmark of NPC1. In this study we found an accumulation of cholesterol in fibroblasts of a NPC1 patient, derived hiPSCs, and neural progenitor cells, but not in cells derived from fibroblasts of a healthy individual. These findings were quantified by the Amplex Red assay, demonstrating a significantly elevated cholesterol level in cells derived from fibroblasts of a NPC1 patient.
We generated a neuronal model based on induced pluripotent stem cells derived from patient fibroblasts, providing a human in vitro model to study the pathogenic mechanisms of NPC1 disease.
尼曼-匹克 C1 型病（NPC1）是一种罕见的进行性神经退行性疾病，由 NPC1 基因突变引起。在这种溶酶体贮积病中，几种脂质（如胆固醇）的细胞内运输和隔离严重受损，导致晚期内体和溶酶体中脂质的积累。该疾病的神经表现是由中枢神经系统的功能障碍和细胞死亡引起的。已经使用了几种动物模型来分析受损的途径。然而，潜在的发病机制仍不完全清楚，并且这些模型不能反映人类的遗传变异性。因此，使用患者特异性诱导多能干细胞的人类模型提供了一种很有前途的方法。
我们通过逆转录病毒转导 Oct4、Klf4、Sox2 和 c-Myc 将 NPC1 患者和健康对照者的人成纤维细胞重编程为诱导多能干细胞（hiPSCs）。通过免疫细胞化学分析对获得的人诱导多能干细胞（hiPSCs）进行了表征。生成神经祖细胞，并进行膜片钳记录以对衍生神经元细胞进行功能分析。使用 filipin 染色和 Amplex Red 测定法来证明和定量胆固醇的积累。
hiPSCs 表达了不同的干细胞标记物，例如 Nanog、Tra-1-81 和 SSEA4。通过胚胎体测定，细胞分化为所有三个胚层的细胞，并在免疫缺陷小鼠中诱导畸胎瘤，证明了它们的多能性。此外，还衍生出神经祖细胞并分化为功能性神经元细胞。膜片钳记录显示电压依赖性通道、自发性动作电位和突触后电流。不同组织中胆固醇的积累是 NPC1 的主要标志。在这项研究中，我们发现 NPC1 患者的成纤维细胞、衍生的 hiPSCs 和神经祖细胞中存在胆固醇的积累，但健康个体的成纤维细胞中没有。这些发现通过 Amplex Red 测定法进行了量化，证明 NPC1 患者成纤维细胞衍生细胞中的胆固醇水平显著升高。
我们基于源自患者成纤维细胞的诱导多能干细胞生成了神经元模型，为研究 NPC1 疾病的发病机制提供了一种人类体外模型。
