RESUMEN
Here, we describe a protocol for a microcarrier (MC)-based, large-scale generation and cryopreservation of human-induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes. We also detail steps to isolate these populations with a high degree of purity. Finally, we describe how to cryopreserve these cell types while maintaining high levels of viability and preserving cellular function post-thaw. For complete details on the use and execution of this protocol, please refer to Brookhouser et al. (2021).
Asunto(s)
Células Madre Pluripotentes Inducidas , Astrocitos , Diferenciación Celular , Células Cultivadas , Humanos , NeuronasRESUMEN
Genome-wide association studies (GWAS) have identified polymorphism in the Apolipoprotein E gene (APOE) to be the most prominent risk factor for Alzheimer's disease (AD). Compared to individuals homozygous for the APOE3 variant, individuals with the APOE4 variant have a significantly elevated risk of AD. On the other hand, longitudinal studies have shown that the presence of the APOE2 variant reduces the lifetime risk of developing AD by 40 percent. While there has been significant research that has identified the risk-inducing effects of APOE4, the underlying mechanisms by which APOE2 influences AD onset and progression have not been extensively explored. In this study, we utilize an isogenic human induced pluripotent stem cell (hiPSC)-based system to demonstrate that conversion of APOE3 to APOE2 greatly reduced the production of amyloid-beta (Aß) peptides in hiPSC-derived neural cultures. Mechanistically, analysis of pure populations of neurons and astrocytes derived from these neural cultures revealed that mitigating effects of APOE2 are mediated by cell autonomous and non-autonomous effects. In particular, we demonstrated the reduction in Aß is potentially driven by a mechanism related to non-amyloidogenic processing of amyloid precursor protein (APP), suggesting a gain of the protective function of the APOE2 variant. Together, this study provides insights into the risk-modifying effects associated with the APOE2 allele and establishes a platform to probe the mechanisms by which APOE2 enhances neuroprotection against AD.
Asunto(s)
Enfermedad de Alzheimer , Apolipoproteínas E/genética , Células Madre Pluripotentes Inducidas , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides , Apolipoproteína E2/genética , Apolipoproteína E4/genética , Estudio de Asociación del Genoma Completo , Humanos , FenotipoRESUMEN
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-ß (Aß), 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.
Asunto(s)
Enfermedades Neurodegenerativas , Células Madre Pluripotentes , Astrocitos , Diferenciación Celular , Humanos , PéptidosRESUMEN
Although the biochemical and pathological hallmarks of Alzheimer's disease (AD), such as axonal transport defects, synaptic loss, and selective neuronal death, are well characterized, the underlying mechanisms that cause AD are largely unknown, thereby making it difficult to design effective therapeutic interventions. Genome-wide association studies (GWAS) studies have identified several factors associated with increased AD risk. Of these genetic factors, polymorphisms in the Apolipoprotein E (APOE) gene are the strongest and most prevalent. While it has been established that the ApoE protein modulates the formation of amyloid plaques and neurofibrillary tangles, the precise molecular mechanisms by which various ApoE isoforms enhance or mitigate AD onset and progression in aging adults are yet to be elucidated. Advances in cellular reprogramming to generate disease-in-a-dish models now provide a simplified and accessible system that complements animal and primary cell models to study ApoE in the context of AD. In this review, we will describe the use and manipulation of human induced pluripotent stem cells (hiPSCs) in dissecting the interaction between ApoE and AD. First, we will provide an overview of the proposed roles that ApoE plays in modulating pathophysiology of AD. Next, we will summarize the recent studies that have employed hiPSCs to model familial and sporadic AD. Lastly, we will speculate on how current advances in genome editing technologies and organoid culture systems can be used to improve hiPSC-based tools to investigate ApoE-dependent modulation of AD onset and progression.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Apolipoproteínas E/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Apolipoproteína E4/metabolismo , Apolipoproteínas E/genética , Estudio de Asociación del Genoma Completo , HumanosRESUMEN
In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs. Specifically, we describe the emergence of technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 that can be used to edit the genome at precise locations, and discuss the strengths and weaknesses of each of these technologies. In addition, we present the current applications of these technologies in elucidating the mechanisms of human development and disease, developing novel and effective therapeutic molecules, and engineering cell-based therapies. Finally, we discuss the emerging technological advances in targeted gene editing methods.