Refining iPSC-Based Spinal Cord Model Systems by Fabricating Developmentally Programmed Extracellular Matrix Cues

ABSTRACT Cellular reprogramming and induced pluripotent stem cell (iPSC) technologies have provided unprecedented access to the human central nervous system (CNS). They have enabled the assembly of models for the investigation of neurodevelopment and neurological disease mechanisms, which have led to significant

advancements in our understanding of these processes. However, culturing iPSC-derived neurons in vitro remains technically challenging. Neurons grown in cell autonomous systems exhibit insufficient levels of morphological maturation, synaptic connectivity, metabolic function, and electrophysiological activity, while

transcriptional analyses suggest they resemble neurons of late embryonic to early postnatal stages hindering the study of adult-onset neurodegenerative diseases. We hypothesize that the lack of a physiological, developmentally appropriate extracellular matrix (ECM) platform is a major contributor to these limitations. The

ECM is an intricately organized intercellular scaffold of secreted proteins and complex sugars that configures spatiotemporal microenvironments throughout the CNS. It provides critical structural support to neurons, astrocytes, and glial cells, serves as a reservoir for soluble factors, and mediates cellular signaling. Through

these actions, the ECM can modulate neuronal development, maturation, and aging. However, the temporal diversity and functional effects of the matrisome, defined as the ensemble of the ECM and ECM-associated proteins, in the CNS is poorly characterized. As a result, the design of in vitro platforms for culturing iPSC-derived

neurons that truly recapitulate the physiological ECM is impossible. Here, we will refine iPSC-neuronal model systems by providing developmentally appropriate ECM cues. In Aim 1 we will utilize biochemical purification and quantitative mass spectrometry (MS)-based proteomics to define the composition and nature of remodeling

of the in vivo matrisome from extracted mouse spinal cords at 3 developmental stages (postnatal, adult, old). In Aim 2 we will leverage our combined expertise in iPSC technologies and biomaterials to establish ECM mimetic matrices that can recapitulate the architecture and modulatory activity of the physiological matrisome to facilitate

the maturation and aging of stem cell derived spinal motor neurons in vitro. In Aim 3 we will improve the development and reproducibility of 3-D spinal organoids by growing them in structured scaffolds with incorporated candidate ECM cues. Our proposed aims will shed light into the contribution of the temporal

matrisome on neuronal diversity, development and function and facilitate the establishment of physiological iPSC-based spinal cord model systems.