Instructive starPEG-Heparin biohybrid 3D cultures for modeling human neural stem cell plasticity, neurogenesis, and neurodegeneration

Three-dimensional models of human neural development and neurodegeneration are crucial when exploring stem-cell-based regenerative therapies in a tissue-mimetic manner. However, existing 3D culture systems are not sufficient to model the inherent plasticity of NSCs due to their ill-defined composition and lack of controllability of the physical properties. Adapting a glycosaminoglycan-based, cell-responsive hydrogel platform, we stimulated primary and induced human neural stem cells (NSCs) to manifest neurogenic plasticity and form extensive neuronal networks in vitro. The 3D cultures exhibited neurotransmitter responsiveness, electrophysiological activity, and tissue-specific extracellular matrix (ECM) deposition. By whole transcriptome sequencing, we identified that 3D cultures express mature neuronal markers, and reflect the in vivo make-up of mature cortical neurons compared to 2D cultures. Thus, our data suggest that our established 3D hydrogel culture supports the tissue-mimetic maturation of human neurons. We also exemplarily modeled neurodegenerative conditions by treating the cultures with Aβ42 peptide and observed the known human pathological effects of Alzheimer’s disease including reduced NSC proliferation, impaired neuronal network formation, synaptic loss and failure in ECM deposition as well as elevated Tau hyperphosphorylation and formation of neurofibrillary tangles. We determined the changes in transcriptomes of primary and induced NSC-derived neurons after Aβ42, providing a useful resource for further studies. Thus, our hydrogel-based human cortical 3D cell culture is a powerful platform for studying various aspects of neural development and neurodegeneration, as exemplified for Aβ42 toxicity and neurogenic stem cell plasticity.SignificanceNeural stem cells (NSC) are reservoir for new neurons in human brains, yet they fail to form neurons after neurodegeneration. Therefore, understanding the potential use of NSCs for stem cell-based regenerative therapies requires tissue-mimetic humanized experimental systems. We report the adaptation of a 3D bio-instructive hydrogel culture system where human NSCs form neurons that later form networks in a controlled microenvironment. We also modeled neurodegenerative toxicity by using Amyloid-beta4 peptide, a hallmark of Alzheimer’s disease, observed phenotypes reminiscent of human brains, and determined the global gene expression changes during development and degeneration of neurons. Thus, our reductionist humanized culture model will be an important tool to address NSC plasticity, neurogenicity, and network formation in health and disease.