SCAN:Novel human central nervous system in vitro models for preclinical research

ITQB Scan seminar

Title: Novel human central nervous system in vitro models for preclinical research
 

Speaker: Catarina Brito

Affiliation: Assistant Researcher at Cell Bioprocesses Laboratory

Abstract:

Central Nervous System (CNS) disorders, such as neurodegenerative diseases, remain a formidable challenge for the development of new and efficient therapies. Gene therapy approaches are promising alternatives that can provide specific targeting and prolonged length of action, treating the causes rather than the symptoms. Hampering the fast translation of gene therapy-based treatments to the clinic is the lack of valid and reliable preclinical models that can contribute to evaluate feasibility and safety. 

Preclinical research has traditionally relied on 2D in vitro cell models, that fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity, etc) and genetically engineered animal models, which often diverge considerably from the human phenotype (developmental, anatomic and physiological). Human 3D in vitro models are useful complementary tools towards more accurate evaluation of drug candidates in preclinical stages, as they present an intermediate degree of complexity between the traditional 2D monolayer culture conditions and the brain.

We have been focusing on the development of robust methodologies for the generation of 3D in vitro models of the human CNS based on stirred culture systems and using human neural stem cells (hNSC) as a scalable supply of differentiated neural-subtype in reproducible ratios. Detailed cell characterization of differentiated neurospheres along culture time includes using spinning disk confocal microscopy, field emission scan electron microscopy (FESEM), transmission electron microscopy (TEM), qRT-PCR and Western Blot.

The feasibility of using this novel 3D model to address viral vector safety was addressed using canine adenovirus type 2 (CAV-2) viral vectors, good candidate for CNS gene therapy applications due to their high cloning capacity, long-term transgene expression and low immunogenicity and tropism towards neurons. The perturbation caused by CAV-2 vectors carrying eGFP reporter gene in human CNS cells was evaluated, aiming at predicting adverse effects.

These model systems constitute a practical and versatile new in vitro approach for preclinical research of human CNS disorders and are expected to increase its relevance. Furthermore, the developed culture strategy may be extended to other sources of human neural stem cells, such as human pluripotent stem cells, including patient-derived induced pluripotent stem cells, broadening the applicability of these models even further.