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Studying Human Neurological Disorders Using Induced Pluripotent Stem Cells: From 2D Monolayer to 3D Organoid and Blood Brain Barrier Models

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ABSTRACT

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 these diseases and the interactions between different brain cell types is essential for the development of new therapeutics. Induced pluripotent stem cells (iPSCs) are invaluable tools for neurological disease modeling, as they have unlimited self‐renewal and differentiation capacity. Mounting evidence shows: (i) various brain cells can be generated from iPSCs in two‐dimensional (2D) monolayer cultures; and (ii) further advances in 3D culture systems have led to the differentiation of iPSCs into organoids with multiple brain cell types and specific brain regions. These 3D organoids have gained widespread attention as in vitro tools to recapitulate complex features of the brain, and (iii) complex interactions between iPSC‐derived brain cell types can recapitulate physiological and pathological conditions of blood‐brain barrier (BBB). As iPSCs can be generated from diverse patient populations, researchers have effectively applied 2D, 3D, and BBB models to recapitulate genetically complex neurological disorders and reveal novel insights into molecular and genetic mechanisms of neurological disorders. In this review, we describe recent progress in the generation of 2D, 3D, and BBB models from iPSCs and further discuss their limitations, advantages, and future ventures. This review also covers the current status of applications of 2D, 3D, and BBB models in drug screening, precision medicine, and modeling a wide range of neurological diseases (e.g., neurodegenerative diseases, neurodevelopmental disorders, brain injury, and neuropsychiatric disorders). © 2019 American Physiological Society. Compr Physiol 9:565‐611, 2019.

Figure 1. Figure 1. Schematic representation of induced pluripotent stem cell (iPSC) generation and application. Many somatic cells (e.g., skin fibroblasts, blood cells, and urine cells) can reprogram into iPSCs, and differentiated into neuronal cell types of interest in both a two‐dimensional (2D) monolayer culture and three‐dimensional (3D) brain organoids, which can be used for modeling human brain disease, elucidating underlying molecular and genetic mechanisms, high‐throughput drug screening, precision medicine, and tissue regeneration. Scale bars without labels: 20 µm
Figure 2. Figure 2. Modeling the blood‐brain barrier (BBB). iPSCs can be differentiated into several cell types of the BBB, including brain microvascular endothelial cells (BMECs), neurons, astrocytes, and pericytes. In vivo, the BBB is comprised of BMECs that form the walls of the blood vessels and are supported by pericytes, astrocytes, neurons, and additional cell types. Current approaches utilized to model the BBB include transwell models, microfluidic and tissue engineering approaches and potentially cell aggregates.
Figure 3. Figure 3. The summarized reported various brain cells generated from iPSCs in two‐dimensional monolayer cultures and their application in the brain physiology and disease modeling.
Figure 4. Figure 4. Tissue sections of 8‐week‐old cerebral organoid generated from human iPSCs. (A) In this whole section of cerebral organoid, red signals represent microtubule associated protein 2 (MAP2)‐positive neurons, green are pax6‐positive neuroepithelial progenitor cells, and nuclei are blue. Scale bar: 500 µm. In the area marked indicated by the yellow arrow, neural stem cells (green) are located in apical side and neurons located in the basal side, suggesting that neurons are differentiated from neural stem cells and migrate from the basal toward the apical side. (B) The red signal represents MAP2‐positive neurons, the green signal represents Synapsin1‐positive synapses between neurons and the blue marks cell nuclei. Scale bar: 50 µm. (C) S100β‐positive astrocytes are shown in green interspersed between MAP2‐positive neurons in red. Scale bar: 5 µm. Blue indicates cell nuclei.


Figure 1. Schematic representation of induced pluripotent stem cell (iPSC) generation and application. Many somatic cells (e.g., skin fibroblasts, blood cells, and urine cells) can reprogram into iPSCs, and differentiated into neuronal cell types of interest in both a two‐dimensional (2D) monolayer culture and three‐dimensional (3D) brain organoids, which can be used for modeling human brain disease, elucidating underlying molecular and genetic mechanisms, high‐throughput drug screening, precision medicine, and tissue regeneration. Scale bars without labels: 20 µm


Figure 2. Modeling the blood‐brain barrier (BBB). iPSCs can be differentiated into several cell types of the BBB, including brain microvascular endothelial cells (BMECs), neurons, astrocytes, and pericytes. In vivo, the BBB is comprised of BMECs that form the walls of the blood vessels and are supported by pericytes, astrocytes, neurons, and additional cell types. Current approaches utilized to model the BBB include transwell models, microfluidic and tissue engineering approaches and potentially cell aggregates.


Figure 3. The summarized reported various brain cells generated from iPSCs in two‐dimensional monolayer cultures and their application in the brain physiology and disease modeling.


Figure 4. Tissue sections of 8‐week‐old cerebral organoid generated from human iPSCs. (A) In this whole section of cerebral organoid, red signals represent microtubule associated protein 2 (MAP2)‐positive neurons, green are pax6‐positive neuroepithelial progenitor cells, and nuclei are blue. Scale bar: 500 µm. In the area marked indicated by the yellow arrow, neural stem cells (green) are located in apical side and neurons located in the basal side, suggesting that neurons are differentiated from neural stem cells and migrate from the basal toward the apical side. (B) The red signal represents MAP2‐positive neurons, the green signal represents Synapsin1‐positive synapses between neurons and the blue marks cell nuclei. Scale bar: 50 µm. (C) S100β‐positive astrocytes are shown in green interspersed between MAP2‐positive neurons in red. Scale bar: 5 µm. Blue indicates cell nuclei.

 

Teaching Material

S. Logan, T. Arzua, S. G. Canfield, E. R. Seminary, S. L. Sison, A. D. Ebert, X. Bai. Studying Human Neurological Disorders Using Induced Pluripotent Stem Cells: From 2D Monolayer to 3D Organoid and Blood Brain Barrier Models. Compr Physiol 9: 2019, 565–611.

Didactic Synopsis

Major Teaching Points:

  • Induced pluripotent stem cells (iPSCs) are able to differentiate into different types of neural cells (e.g., neurons, astrocytes, and oligodendrocytes) by being cultured in chemically defined induction media in 2-dimensional (2D) monolayer cultures.
  • iPSC-derived 3-dimensional (3D) cortical organoids are composed of multiple neural cell types and exhibit defined brain regions. Cortical organoids have received widespread attention as in vitro tools to recapitulate function, architecture, and geometric features of human brain tissues and offer an unprecedented opportunity to study complex human diseases that affect multiple cell types, their interactions, and the function of neuronal circuits.
  • iPSC-derived astrocytes, endothelial cells, neurons, and pericytes have been utilized to construct the blood-brain barrier (BBB) in culture dishes. Human iPSC-derived BBB replicates key features of the BBB seen in vivo and enables new mechanistic investigations of BBB functions in neurological diseases and drug screening.
  • 2D cell models, BBB models, and 3D organoids have different advantages and limitations for studying neurological diseases.
  • Human iPSC disease modeling has key advantages compared to animal models such as providing a complicated genetic signature of patients and unlimited cell resource. Human iPSC-derived 2D,3D, and BBB systems have been used for modeling various neurological disorders to study the pathological phenotypes and mechanisms. Specifically, patient-specific iPSC-derived neural cells provide a promising human model for precision medicine research in dissecting genetic contribution to disease development, testing the efficiency and toxicity of drugs, and developing new therapeutics for neurological disorders.
  • iPSC-derived 2D cell models 3D organoids and BBB models, have been used for in vitro modeling of neurodegenerative diseases [e.g., Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and traumatic brain injury (TBI), Huntington's disease, neurodevelopmental disorders (e.g., cytomegalovirus (CMV) infection, Zika virus infection, autism, Rett syndrome, and Allan-Herndon-Dudley syndrome (AHDS), and neuropsychiatric disorders (e.g., schizophrenia and bipolar disorder)]. The findings have provided novel insights into molecular and genetic mechanisms of brain diseases.
  • Techniques in iPSCs, gene editing, and patient-specific cells are at the forefront of approaches towards personalized medicine initiatives. Gene editing allows for disease modeling and obtaining cells from patients suffering from conditions allows for the study of response to therapeutics and phenotypic analysis.
  • Limitations of iPSC-based models for neurological disorders (e.g., immature features of differentiated neural cells and lack of vascularization) should be taken into consideration in future studies. Developments in iPSC technology and other rapid advances in cellular, molecular and developmental neurobiology will be the future driving forces to accelerate development of more clinically relevant human iPSC models and new treatments for people with neurological disorders.

Didactic Legends

The figures—in a freely downloadable PowerPoint format—can be found on the Images tab along with the formal legends published in the article. The following legends to the same figures are written to be useful for teaching.

Figure 1 Teaching Points: Knowing that human induced pluripotent stem cells (iPSCs) can be generated from many different somatic such as urine. iPSCs can be differentiated into neuronal cell types of interest in both a 2-dimensional (2D) monolayer culture, 3-dimensional (3D) brain organoids and blood brain barrier, which have wide applications in biomedical fields such as modeling human brain disease, elucidating underlying molecular and genetic mechanisms, high-throughput drug screening for efficacy/toxicity, precision medicine, and tissue regeneration. Scale bar: 20 µm

Figure 2 Teaching Points: This figure illustrates the summarized reported various brain cells generated from iPSCs in 2-dimensional monolayer cultures and their application in the brain physiology and disease modeling.

Figure 3 Teaching Points: In vivo, the blood-brain barrier (BBB) is comprised of brain microvascular endothelial cells (BMECs) that form the walls of the blood vessels and are supported by pericytes, astrocytes, neurons, and additional cell types such as neurons. BBB can be modeled using iPSC-derived BMECs, pericytes, astrocytes, and neurons. Current approaches utilized to model the BBB include transwell models, microfluidic and tissue engineering approaches, and cell aggregates.

Figure 4 Teaching Points: This figure illustrates the 8-week old cerebral organoid generated from human iPSCs. 3D cerebral organoids include various types of brain cells: neural stem cells, neurons with synapse signals, and astrocytes. In addition, the 3D cerebral organoids development process recapitulates some human brain developmental patterns such as neurons (red) differentiate from neural stem cells (green) that are located apical sides and migrates from apical toward basal sides.

 


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How to Cite

Sarah Logan, Thiago Arzua, Scott G. Canfield, Emily R. Seminary, Samantha L. Sison, Allison D. Ebert, Xiaowen Bai. Studying Human Neurological Disorders Using Induced Pluripotent Stem Cells: From 2D Monolayer to 3D Organoid and Blood Brain Barrier Models. Compr Physiol 2019, 9: 565-611. doi: 10.1002/cphy.c180025