Modelling human CNS injury with human neural stem cells in 2- and 3-Dimensional cultures

Abstract

The adult human central nervous system (CNS) has very limited regenerative capability, and injury at the cellular and molecular level cannot be studied in vivo. Modelling neural damage in human systems is crucial to identifying species-specific responses to injury and potentially neurotoxic compounds leading to development of more effective neuroprotective agents. Hence we developed human neural stem cell (hNSC) 3-dimensional (3D) cultures and tested their potential for modelling neural insults, including hypoxic-ischaemic and Ca 2+ -dependent injury. Standard 3D conditions for rodent cells support neuroblastoma lines used as human CNS models, but not hNSCs, but in all cases changes in culture architecture alter gene expression. Importantly, response to damage differs in 2D and 3D cultures and this is not due to reduced drug accessibility. Together, this study highlights the impact of culture cytoarchitecture on hNSC phenotype and damage response, indicating that 3D models may be better predictors of in vivo response to damage and compound toxicity.In light of the practical and ethical limitations of human research, animal models provide fundamental insight into the central nervous system (CNS) development, injury, disease, as well as the possibility of studying and screening putative therapeutic compounds. However, there is an increasing appreciation of the limitations of animal research, which often fails to reliably predict successful human clinical trials. Key examples of this are hypoxia-ischemia either in newborns (1-8 cases per 1000 births in the Western world) or adults (cerebral stroke) and traumatic injuries to the CNS 1-3 . While animal models have significantly furthered understanding of the mechanisms of injury and repair, and various neuroprotective agents have proven effective in animal injury models, this has not translated into effective treatments in humans 4-6 . Such failures to translate animal studies to successful human clinical trials are often attributed to problems with methodological and study design. However, species-specific differences in drug processing, genetic interactions and molecular mechanisms of action can be argued to be fundamental to therapeutic translation. Further studies on the cell ular mechanisms of human CNS injury and repair are therefore needed in order to develop better therapeutic strategies and bridge the gap between animal models and human clinical trials 7,8 .Three dimensional (3D) culture models have been rapidly emerging as a means to study human cells in vitro. While still in many respects rather challenging, because more complex to maintain and analyse, they reduce the strain and artificial responses which cells must undergo in order to adapt to the flat, stiff surfaces of 2D monolayer culture. A 3D architecture provides a more tissue-like environment in which cell-cell and cell-matrix interact in all dimensions and cells have more biologically relevant exposure to diffusible factors 9,10 . Whereas the use of iPSCs (induced pluripotent stem cells) derived f...

Dystrophin deficiency affects human astrocyte properties and response to damage

“…For 3D cultures, cells were mixed in hydrogels consisting of 1 mg/ml collagen I (Rat Tail High Concentration, Corning) and 2 mg/ml Matrigel (Corning) as described elsewhere (Vagaska et al, 2020) and allowed to polymerize for 40 min. In brief, a cold collagen I solution at pH to 7.4 was added to the Matrigel at 4°C before mixing the hydrogel with the cell suspension and inducing polymerization at 37°C for 30 min.…”

Bioorthogonally Cross-Linked Hyaluronan-Laminin Hydrogels for 3D Neuronal Cell Culture and Biofabrication

“…Our findings support previous data showing that Matrigel can allow the formation of complex neuronal networks. [81] Depending on the size and format of the 3D-hydrogel being stained, lengthy immunostaining protocols are needed, compared to 2D cultures, to provide enough diffusion time for antibodies to penetrate the gel and bind to the cells. With Matrigel, we found it difficult to avoid unspecific binding and high background noise even with repeated and longer washing steps.…”

“…When compared to the undifferentiated state of lt-NES, we observe that all conditions had significant upregulation of DCX (Figure S6, Supporting Information), which is in line with previous studies where neuroepithelial stem cells show expression of DCX after 7 days of neuronal differentiation. [81] The later neuronal marker Tubulin Beta 3 Class III (TUBB3) shows a slight upregulation (1-1.5 times) compared to undifferentiated lt-NES (Figure S6, Supporting Information), where Matrigel again had the highest level of upregulation. However, when comparing expression between the different hydrogel conditions (Figure 5b), we do not see any significant up or downregulation when adding LN or LN-az.…”

“…Our findings support previous data showing that Matrigel can allow the formation of complex neuronal networks. [81] . CC-BY 4.0 International license perpetuity.…”

Modelling the central nervous system: tissue engineering of the cellular microenvironment

“…In vitro models offer greater applicability to human tissues [ 5 ]; however, existing culture techniques are predominantly carried out as 2D cultures and are incapable of recreating true three-dimensional (3D) tissue complexity. Such reductionist approaches are deemed unable to reliably predict clinical outcomes in humans [ 3 , 13 ]. 3D culture techniques enable creation of in vitro models of the CNS with superior biological relevance, with an additional dimension of features that may present novel phenotypic markers of disease [ 14 ].…”