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

Abstract

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.

EMT-induced cell mechanical changes enhance mitotic rounding strength

“…In situ assembling biohybrid multiarmed poly(ethylene glycol) (starPEG)-heparin hydrogels, prepared as previously reported 56 , were used for embedding cells to grow into speroids. In brief, cells were detached from the tissue flasks and resuspended in PBS.…”

“…After equilibrating them at room temperature for 1 hour, gels were mounted onto glass object slides using Cell-Tak TM (Thermofisher). Gel stiffness was probed by AFM indentation 18,55,56 . Cantilevers (arrow T1, Nanoworld), that had been modified with polystyrene beads of 10 μm diameter (Microparticles GmbH) using epoxy glue (Araldite), were calibrated using the thermal noise method implemented in the AFM software and probed at room temperature in PBS using a speed of 5 μm/sec and a relative force setpoint of 2.5 nN.…”

Interleukin-4 restores neurogenic plasticity of the primary human neural stem cells through suppression of Kynurenic acid production upon Amyloid-beta42 toxicity

“…Previously, we have shown that in 3D gel cultures, primary human NSC plasticity, neurogenic ability, and neuronal network forming capacity are reduced by Aβ42 (Papadimitriou et al, 2017), and IL4 has a positive effect on NSC proliferation in an Aβ42-based Alzheimer’s model of adult zebrafish brain (Bhattarai et al, 2016). Therefore, to determine whether IL4 treatment would have an effect on the reduction of NSC plasticity, neurogenic properties and network formation of the neurons, we cultured Aβ42-treated cells in the presence of hydrogel-administered IL4, and compared it to control and Aβ42-treated cells without IL4 (Figure 2A-F).…”

“…StarPEG-heparin hydrogels were generated as previously described (Maitz et al, 2013; Wieduwild et al, 2013) and (Papadimitriou et al, 2017). Amyloid peptides were synthesized as previously described (Bhattarai et al, 2017; Bhattarai et al, 2016; Kizil et al, 2015a; Wieduwild et al, 2013).…”

“…Sample preparation, RNA isolation, library preparation, next-generation sequencing, data analyses are performed as described before (Papadimitriou et al, 2017).…”