In this work, we developed a microcarrier (MC)-based culture system that allows for the expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells (hNPCs) under defined conditions. In turn, this MC approach was implemented in a rotating wall vessel (RWV) bioreactor for the large-scale expansion and neuronal differentiation of hNPCs. This work is of significance as it overcomes current limitations of conventional two dimensional (2-D) culture systems to enable the generation of hNPCs and their neuronal derivatives in quantities required for downstream applications in disease modeling, drug screening, and regenerative medicine.

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 (Aβ) 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 Aβ 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.

Despite therapeutic advances, neurodegenerative diseases and disorders remain some of the leading causes of mortality and morbidity in the United States. Therefore, cell-based therapies to replace lost or damaged neurons and supporting cells of the central nervous system (CNS) are of great therapeutic interest. To that end, human pluripotent stem cells (hPSC) derived neural progenitor cells (hNPCs) and their neuronal derivatives could provide the cellular ‘raw material’ needed for regenerative medicine therapies for a variety of CNS disorders. In addition, hNPCs derived from patient-specific hPSCs could be used to elucidate the underlying mechanisms of neurodegenerative diseases and identify potential drug candidates. However, the scientific and clinical application of hNPCs requires the development of robust, defined, and scalable substrates for their long-term expansion and neuronal differentiation. In this study, we rationally designed a vitronectin-derived peptide (VDP) that served as an adhesive growth substrate for the long-term expansion of several hNPC lines. Moreover, VDP-coated surfaces allowed for the directed neuronal differentiation of hNPC at levels similar to cells differentiated on traditional extracellular matrix protein-based substrates. Overall, the ability of VDP to support the long-term expansion and directed neuronal differentiation of hNPCs will significantly advance the future translational application of these cells in treating injuries, disorders, and diseases of the CNS.

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, peptidebased 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.

Numerous epidemiological studies have demonstrated that individuals who have sustained a traumatic brain injury (TBI) have an elevated risk for developing Alzheimer’s disease and Alzheimer’s-related dementias (AD/ADRD). Despite these connections, the underlying mechanisms by which TBI induces AD-related pathology, neuronal dysfunction, and cognitive decline have yet to be elucidated. In this review, we will discuss the various in vivo and in vitro models that are being employed to provide more definite mechanistic relationships between TBI-induced mechanical injury and AD-related phenotypes. In particular, we will highlight the strengths and weaknesses of each of these model systems as it relates to advancing the understanding of the mechanisms that lead to TBI-induced AD onset and progression as well as providing platforms to evaluate potential therapies. Finally, we will discuss how emerging methods including the use of human induced pluripotent stem cell (hiPSC)-derived cultures and genome engineering technologies can be employed to generate better models of TBI-induced AD.