Novel human iPSC models of neuroinflammation in neurodegenerative disease and regenerative medicine

Abstract

The importance of neuroinflammation in neurodegenerative diseases is becoming increasingly evident, and, in parallel, human induced pluripotent stem cell (hiPSC) models of physiology and pathology are emerging. Here, we review new advancements in the differentiation of hiPSCs into glial, neural, and blood-brain barrier (BBB) cell types, and the integration of these cells into complex organoids and chimeras. These advancements are relevant for modeling neuroinflammation in the context of prevalent neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). With awareness of current limitations, recent progress in the development and application of various hiPSC-derived models shows potential for aiding the identification of candidate therapeutic targets and immunotherapy approaches.

Keywords: Alzheimer’s disease; Parkinson’s disease; cell differentiation; glia; hiPSC; microglia; multiple sclerosis; neuroinflammation; organoids.

Figure 1.. Human induced pluripotent stem cell (hiPSC)-based models of neuroinflammation in Alzheimer’s disease (AD), Parkinson’s disease (PD), and multiple sclerosis (MS).

AD, PD, and MS pathologies involve dysregulated neuroinflammatory processes (including inflammatory glial states) characterized by the secretion of pro-inflammatory cytokines and complement factors [2], in addition to the generation of reactive oxygen species and compromised brain-barrier integrity [5]. To investigate these neuroinflammatory disease mechanisms, hiPSC can be differentiated to form 2D glial cell cultures, 3D glia-containing neural organoids, brain barrier-specific cell types and structures, and chimeric models formed from xenotransplantation of human cells (including neural precursor cells, NPC) and organoids, into animals.

Figure 2.. Modeling of CNS cells using iPSC-derived 2D and 3D models.

Somatic cells, such as skin fibroblasts and blood cells, can undergo reprogramming into a pluripotent state (iPSC) by transfection of the four “Yamanaka factors” (Oct3/4, Sox2, Klf4, and c-Myc) [6], using viral or nonviral vector transfer systems. Next, iPSC can be differentiated into 2D and 3D cultures using one of two different approaches: (1) adding guiding agents to the culture medium to mimic in vivo developmental signaling stimuli (green arrows); or (2) overexpressing cell fate-determining transcription factors required for each specific lineage commitment (purple arrows). The iPSC can also be differentiated to hematopoietic progenitors and from there to microglia [–14] or to neural progenitor cells [112] and from there to oligodendrocytes [–26], astrocytes [–22], or neurons. Neurons and microglia can be directly differentiated from somatic cells or iPSC [15,86]. Moreover, iPSC or neural progenitor cells can be used to generate 3D models, such as neural organoids (orange arrows) [,–40]. A number of co-cultures of iPSC-derived CNS cells can be reproduced in vitro (dashed arrows) [12,91,93].

Figure 3.. Experimental workflow for generating an in-vitro platform to study neuroinflammation in MS.

MS patients are selected based on their in vivo 3-tesla MRI scan and the presence of chronically inflamed MS lesions (visualized by the presence of a paramagnetic rim on susceptibility-sensitive MRI; magnification in the inset). Further neuropathological characterization of the lesions is performed using multiplex immunofluorescence and single-cell transcriptomics (inset: scale bar 50 μm) [41]. In parallel, somatic cells (i.e., fibroblasts or blood cells) from MS patients are reprogrammed into hiPSC and subsequently differentiated to produce glia-enriched organoids. In addition, CSF from the same patient (represented by a droplet) is collected and characterized in terms of its inflammatory profile (quantification of inflammatory cytokines, chemokines, and immunoglobulin subtypes). Exposure of organoids to patient-derived CSF allows the generation of MS-inflamed, glia-enriched organoids that partially recapitulate in vitro several of the inflammatory and neurodegenerative glia phenotypes (MIMS-iron, inflammatory astrocytes, and immunological OPC, but not MIMS-foamy) that have been observed in autopsy human brain tissues [32,41]. CSF, cerebrospinal fluid; hiPSC, human induced pluripotent stem cells; MIMS, microglia inflamed in MS; MRI, magnetic resonance imaging; MS, multiple sclerosis; OPC, oligodendrocyte precursor cell; T2-FLAIR, T2-weighted fluid-attenuated inversion recovery.

Key figure, Figure 4.. Proposed future directions for human induced pluripotent stem cell (hiPSC)-based models of neuroinflammation in neurodegenerative diseases.

To improve current hiPSC-based models of neuroinflammation, we suggest that researchers focus on validating the biological relevance of these models by thoroughly comparing the transcriptomes of hiPSC-derived cells to human primary cells as represented by the uniform manifold appxomation and projection (UMAP) graph icon, and using this and other methods to further recapitulate the heterogeneity of these cell phenotypes and functions to those found in vivo. Additionally, exposure to disease-specific stimuli, rather than general inflammatory stimuli, will allow future studies to narrow in on relevant pathological mechanisms, and utilizing modern approaches to study genetic perturbations will be similarly valuable. These efforts, paired with the continued use of hiPSC-based models of neuroinflammation, can contribute to the development of brain barrier-penetrating immunotherapies as well as novel regenerative therapies facilitated by transplantation of hiPSC-derived cells and organoids into the human CNS, ultimately advancing care for patients with neurodegenerative diseases including AD, PD, and MS.