Pushing the boundaries of brain organoids to study Alzheimer's disease

Abstract

Progression of Alzheimer's disease (AD) entails deterioration or aberrant function of multiple brain cell types, eventually leading to neurodegeneration and cognitive decline. Defining how complex cell-cell interactions become dysregulated in AD requires novel human cell-based in vitro platforms that could recapitulate the intricate cytoarchitecture and cell diversity of the human brain. Brain organoids (BOs) are 3D self-organizing tissues that partially resemble the human brain architecture and can recapitulate AD-relevant pathology. In this review, we highlight the versatile applications of different types of BOs to model AD pathogenesis, including amyloid-β and tau aggregation, neuroinflammation, myelin breakdown, vascular dysfunction, and other phenotypes, as well as to accelerate therapeutic development for AD.

Keywords: Alzheimer’s disease; Apolipoprotein E4; brain organoids; drug discovery; induced pluripotent stem cells; myelin organoids.

Figure 1:. Generation and applications of isogenic APOE3 and APOE4 brain organoids.

Patient-derived fibroblasts are first obtained from a skin biopsy of a carrier of APOE4. Fibroblasts are then reprogrammed into induced pluripotent stem cells (iPSCs), and a single nucleotide point mutation (cytosine-to-thymine) is introduced using CRISPR/Cas9-based genetic editing, resulting in a missense mutation and exchange of arginine (Arg) for cysteine (Cys), generating the APOE3 variant. Subsequently, unedited APOE4 iPSCs and isogenic edited APOE3 iPSCs are differentiated into brain organoids (BOs) following well-established protocols. The resulting BOs share the same genetic background except for the APOE variant, enabling mechanistic studies of the roles of ApoE isoforms in lipid metabolism, neuroinflammation, cellular stress, and other processes, without the confounding effects of patient-to-patient variation.

Figure 2 (Key Figure):. Strategies for obtaining glial cell-enriched brain organoids.

(A) Long-term culture of neural organoids promotes astrocyte differentiation as radial glia progenitors undergo the neurogenic-to-gliogenic transition. Long-term maintenance of neural organoids can be further improved by slicing spherical organoids and maintaining sliced organoids to allow efficient nutrient and oxygen exchange. Astrocyte-enriched neural organoids can be used to study astrocyte lipid metabolism and astrogliosis in AD, among other roles. (B) Supplying key signaling molecules required for oligodendrocyte progenitor cell (OPC) and oligodendrocyte differentiation, including platelet-derived growth factor AA (PDGF-AA), insulin-like growth factor 1 (IGF-1), and thyroid hormone (T3), promotes OPC and oligodendrocyte differentiation in myelinoids. Patterning myelinoids towards ventral spinal cord identity can further improve myelination. Oligodendrocyte-enriched myelinoids can be used to study the effects of myelination and myelin breakdown in AD. (C) Given that microglia arise from a different germ layer (mesoderm) than do neural tissues (ectoderm), generation of microglia-containing neuroimmune organoids (NIOs) can be achieved by seeding induced pluripotent stem cell (iPSC)-derived microglia into differentiated BOs. To obtain microglia, iPSCs are differentiated into an erythromyeloid progenitor-like intermediate followed by microglia differentiation and maturation in the presence of transforming growth factor β (TGF-β), interleukin 34 (IL-34), and macrophage colony-stimulating factor (M-CSF). Microglia-enriched NIOs can be used to study phagocytosis of toxic debris and microgliosis in AD.

Figure 3:. Human organoid technology for modeling brain vascular systems.

(A) Vascularized BOs can be engineered by transcription factor-driven directed endothelial cell differentiation during BO generation. Alternatively, spheroids containing astrocytes, pericytes, and endothelial cells self-organize into distinct layers that form a blood-brain barrier (BBB)-like structure. Finally, organ-on-a-chip devices combine microfluidics with in vitro cell culture, enabling the desired layering of different BBB cell types to be achieved. (B) Differentiation of choroid plexus organoids (ChPOs) enables modeling of the blood-cerebrospinal fluid (B-CSF) barrier and the production of CSF-like fluid. Furthermore, ChPOs may be used to study the changes in CSF composition during AD progression as well as to uncover AD-relevant CSF biomarkers. (C) Organoid models of brain vascular systems recapitulate key elements of the BBB and the B-CSF barrier. For example, BBB models contain endothelial cells, pericytes, and astrocytic end-feet as well as express genes encoding tight junction and channel proteins, such as aquaporin 4 (AQP4). ChPOs contain secretory epithelium producing CSF-like fluid as well as express genes encoding tight junction proteins, such as claudin 1 (CLDN1), and proteins required for CSF secretion, such as aquaporin 1 (AQP1). Therefore, the BBB and B-CSF barrier models can be used to study various aspects of vascular dysfunction associated with AD, and explore novel directions for AD research, such as the roles of peripheral lipoproteins and ApoE as well as the adaptive immune system in AD progression.