Human Pluripotent Stem Cell-derived Astrocytes Research Articles

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

A Defined and Scalable Peptide-Based Platform for the Generation of Human Pluripotent Stem Cell-Derived Astrocytes.

Astrocytes comprise the most abundant cell type in the central nervous system (CNS) and play critical roles in maintaining neural tissue homeostasis. In addition, astrocyte dysfunction and death has been implicated in numerous neurological disorders such as multiple sclerosis, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and Parkinson’s disease (PD). As such, there is much interest in using human pluripotent stem cell (hPSC)-derived astrocytes for drug screening, disease modeling, and regenerative medicine applications. However, current protocols for generation of astrocytes from hPSCs are limited by the use of undefined xenogeneic components and two-dimensional (2D) culture surfaces, which limits their downstream applications where large-quantities of cells generated under defined conditions are required. Here, we report the use of a completely synthetic, peptide-based substrate that allows for the differentiation of highly pure populations of astrocytes from several independent hPSC lines, including those derived from patients with neurodegenerative disease. This substrate, which we demonstrate is compatible with both conventional 2D culture formats and scalable microcarrier (MC)-based technologies, leads to the generation of cells that express high levels of canonical astrocytic markers as well as display properties characteristic of functionally mature cells including production of apolipoprotein E (ApoE), responsiveness to inflammatory stimuli, ability to take up amyloid-β (Aβ), and appearance of robust calcium transients. Finally, we show that these astrocytes can be cryopreserved without any loss of functionality. In the future, we anticipate that these methods will enable the development of bioprocesses for the production of hPSC-derived astrocytes needed for biomedical research and clinical applications.

Human Stem Cell-derived Aggregates of Forebrain Astroglia Respond to Amyloid Beta Oligomers.

Astrocytes are vital components in neuronal circuitry and there is increasing evidence linking the dysfunction of these cells to a number of central nervous system diseases. Studying the role of these cells in human brain function in the past has been difficult due to limited access to the human brain. In this study, human induced pluripotent stem cells were differentiated into astrospheres using a hybrid plating method, with or without dual SMAD inhibition. The derived cells were assessed for astrocytic markers, brain regional identity, phagocytosis, calcium-transient signaling, reactive oxygen species production, and immune response. Neural degeneration was modeled by stimulation with amyloid-β (Aβ) 42 oligomers. Finally, co-culture was performed for the derived astrospheres with isogenic neurospheres. Results indicate that the derived astroglial cells express astrocyte markers with forebrain dorsal cortical identity, secrete extracellular matrix, and are capable of phagocytosing iron oxide particles and responding to Aβ42 stimulation (higher oxidative stress, higher TNF-α, and IL-6 expression). RNA-sequencing results reveal the distinct transcriptome of the derived cells responding to Aβ42 stimulation for astrocyte markers, chemokines, and brain regional identity. Co-culture experiments show the synaptic activities of neurons and the enhanced neural protection ability of the astroglial cells. This study provides knowledge about the roles of brain astroglial cells, heterotypic cell-cell interactions, and the formation of engineered neuronal synapses in vitro. The implications lie in neurological disease modeling, drug screening, and studying progression of neural degeneration and the role of stem cell microenvironment. Impact Statement Human pluripotent stem cell-derived astrocytes are a powerful tool for disease modeling and drug screening. However, the properties regarding brain regional identity and the immune response to neural degeneration stimulus have not been well characterized. Results of this study indicate that the derived astroglial cells express astrocyte markers with forebrain dorsal cortical identity, secrete extracellular matrix (ECM), and are capable of phagocytosing iron oxide particles and responding to amyloid-β oligomers, showing the distinct transcriptome in astrocyte markers, chemokines, and brain regional identity. This study provides knowledge about the roles of brain astroglial cells, heterotypic cell-cell interactions, and engineering neural tissues in vitro.

Modeling Neurological Disorders with Human Pluripotent Stem Cell-Derived Astrocytes.

Astrocytes play vital roles in neurological disorders. The use of human induced pluripotent stem cell (iPSC)-derived astrocytes provides a chance to explore the contributions of astrocytes in human diseases. Here we review human iPSC-based models for neurological disorders associated with human astrocytes and discuss the points of each model.

Regionally specified human pluripotent stem cell-derived astrocytes exhibit different molecular signatures and functional properties

Astrocytes display diverse morphologies in different regions of the central nervous system. Whether astrocyte diversity is attributable to developmental processes and bears functional consequences, especially in humans, is unknown. RNA-seq of human pluripotent stem cell-derived regional astrocytes revealed distinct transcript profiles, suggesting differential functional properties. This was confirmed by differential calcium signaling as well as effects on neurite growth and blood-brain barrier formation. Distinct transcriptional profiles and functional properties of human astrocytes generated from regionally specified neural progenitors under the same conditions strongly implicate the developmental impact on astrocyte diversity. These findings provide a rationale for renewed examination of regional astrocytes and their role in the pathogenesis of psychiatric and neurological disorders.