Isolation and Culture of Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoid Cells
The advent of human-induced pluripotent stem cell (iPSC)-derived three-dimensional (3D) cerebral organoids provides unprecedented opportunities of modeling human brains in states of health and disorder. Emerging data supports that cerebral organoids allow
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Isolation and Culture of Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoid Cells Yasheng Yan, Thiago Arzua, Sarah Logan, and Xiaowen Bai Abstract The advent of human-induced pluripotent stem cell (iPSC)-derived three-dimensional (3D) cerebral organoids provides unprecedented opportunities of modeling human brains in states of health and disorder. Emerging data supports that cerebral organoids allow for more relevant in vitro systems for studying the human brain system and diseases than the current widely used 2D monolayer cell culture. Thus, the ability to isolate, culture, and maintain human brain cells from cerebral organoids is highly needed, particularly for studies on organoid-derived cell-type-specific signaling and their electrophysiological properties. Here we present a protocol to isolate and culture brain cells from 2-month human iPSC-derived cerebral organoids. The dissociation and plating of cells from organoids takes 3–4 h. The dissociated cells can be maintained in culture for up to at least 3 weeks. Some cells expressed the neuron-specific marker microtubule-associated protein 2 and exhibited spontaneous action potentials. Key words Cerebral organoids, Dissociation, Induced pluripotent stem cells, Neurons
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Introduction Neurological disorders have emerged as a predominant healthcare concern in recent years due to their severe consequences on quality of life and prevalence throughout the world. Understanding the underlying mechanisms of neurological diseases and the contribution of different types of brain cells is essential for the development of new therapeutics. Animals have been widely used in brain research as models to predict human brain response to drugs and environmental stressors. However, inherent species-specific differences in brain physiology and genetics are increasingly acknowledged [1–4]. There is a crucial need for neurological models with higher fidelity to human brains. Human-induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells (e.g., fibroblasts and excreted urine cells) are able to replicate indefinitely and differentiate into virtually every cell type [5–8]. Recently developed human iPSC-derived three-dimensional (3D) cerebral organoids (Fig. 1a) from Dr. Knoblich’s lab allow more relevant in vitro systems for studying the human brain system and diseases than animal models.
Yasheng Yan et al.
Fig. 1 Human-induced pluripotent stem cell-derived 2-month 3D cerebral organoids. (a) Morphology of cerebral organoids. (b) The image of the immunostained cerebral organoid tissue section. Ventricle-like lumens appear within the 3D organoids. The organoids display well-organized elaborate cellular laminar architectures. Neural stem cells and neurons are located in the different layers of the organoid tissues. SOX2-positive neural stem cells (green) are located on the apical side, and neurons (red) are located on the basal side (periphery) of the organoids. Blue are cell nuclei stained with Hoechst 33342
Cerebral organoids can be generated in vitro by growi
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