Effects of 3D culturing conditions on the transcriptomic profile of stem-cell-derived neurons

Abstract

Understanding neurological diseases requires tractable genetic systems. Engineered 3D neural tissues are an attractive choice, but how the cellular transcriptomic profiles in these tissues are affected by the encapsulating materials and are related to the human-brain transcriptome is not well understood. Here, we report the characterization of the effects of culturing conditions on the transcriptomic profiles of induced neuronal cells, as well as a method for the rapid generation of 3D co-cultures of neuronal and astrocytic cells from the same pool of human embryonic stem cells. By comparing the gene-expression profiles of neuronal cells in culture conditions relevant to the developing human brain, we found that modifying the degree of crosslinking of composite hydrogels can tune expression patterns so they correlate with those of specific brain regions and developmental stages. Moreover, by using single-cell sequencing, we show that our engineered tissues recapitulate transcriptional patterns of cell types in the human brain. The analysis of culturing conditions will inform the development of 3D neural tissues for use as tractable models of brain diseases.

Screening of Hydrogels for Human Pluripotent Stem Cell–Derived Neural Cells: Hyaluronan‐Polyvinyl Alcohol‐Collagen‐Based Interpenetrating Polymer Network Provides an Improved Hydrogel Scaffold

“…We conclude that the use of more complex analysis methods, such as multiparameter analysis and PCA, can allow more relevant information to be obtained with sufficient time, in line with a recent study . Here, Col1 alone was the most supportive scaffold, although it was mechanically unstable.…”

“…Combining information from different indexes reveals results from a wider perspective, as shown recently . According to the evaluated indexes during the experiments, gel behavior differed between hydrogels both in the case of gel performance properties (gel performance index, Table S3, Supporting Information) and supportiveness of neurite spreading (neurite spreading index, Table S2, Supporting Information) (Figure B).…”

“…The crucial aspect of the material selected for these scaffolds is that it allows an interaction between cells and the material that is similar to that in vivo between cells and the extracellular matrix (ECM). Different material properties, including mechanical properties, such as stiffness, material chemistry, availability for binding sites, and porosity, affect this interaction …”

Using stem cell–derived neurons in drug screening for neurological diseases

“…Recent advances in protocols to culture brain organoids, or 3D co-cultures of brain cells, may further improve the physiological relevance of in vitro models. For example recent studies have shown that 3D co-cultures rapidly produce synapses (Krencik et al, 2017), and recapitulate transcriptional patterns of cell types in the human brain (Tekin et al, 2018). 3D co-culture has recently been used to assess toxicology (Sirenko et al, 2018) and can reveal toxic effects on processes such as cell migration which cannot be determined in two dimensional culture (Hellwig et al, 2018).…”

High-content interrogation of human induced pluripotent stem cell-derived cortical organoid platforms

“…Several protocols have been developed to convey regional identities by the addition of patterning factors. Studies also have shown that neurons in 3D cultures are more electrically robust, and express more genes involved in neurological processes [5][6][7] . Many different methods for generating 3D cultures have emerged, including spheroid cultures, spinning bioreactors, microfluidic systems, and biopolymer scaffolds 2 .…”