The effect of different scaffolds on the differentiation of a single cell type: 2D and 3D scaffolds on neural progenitor cells differentiation.
A longstanding difference between in vitro and in vivocell growth conditions is the dimensionality difference between the two.In vitro experiments have traditionally used two-dimensional cell monolayers (discounting recent organoid developments), while cells in live organisms develop in a three-dimensional setting. The dimensionality of a cell’s microenvironment can have profound effects on the function and differentiation of said cell (Willerth & Sakiyama-Elbert, 2019). Transcriptomic responses can be documented using high-throughput techniques like microarray and RNA sequencing.
In 2011, Lai et al used 2D and 3D porous polystyrene scaffolds to assess and compare the effect of dimensionality on neural progenitor cell (NPC) differentiation (Lai et al., 2011). NPCs were maintained in neural basal media supplemented with penicillin, L-glutamine, recombinant human leukemia inhibitory factor, basic fibroblast growth factor (bFGF) and B-27 (Lai et al., 2011). Later on, bFGF was removed from above mentioned factors to start differentiation process 25. NPCs cultured in the presence of the 2D substrate showed significant transcriptomic remodeling with 890 DEGs, with 439 of these genes up-regulated (FC>2 and p-value <0.05). The differentiated NPCs were clearly segregated from undifferentiated cells (Fig. 1a). The most statistically significant upregulated genes were those related to the central nervous system (CNS), including nervous system development, axon guidance, and axonogenesis. (Fig. 1b, Table S1). The same type of analysis with the same criteria (FC>2 and p-value <0.05) for cells grown in a 3D scaffold led us to find 1082 DEGs, most of which were upregulated. The differentiated cells in presence of the 3D substrate also were separated from undifferentiated cells (Fig1a). The analysis of biological processes similarly showed neuronal differentiation related terms as the most significantly upregulated terms (Fig. 1c, Table S2). We then compared the list of DEGs between each scaffold dimension to elucidate the differences between cultures. The results of this comparison revealed a high level of transcriptome similarity between culturing neural progenitor cells in 2D and 3D scaffolds (Fig. 1d). 52% of DEGs in the 2D scaffold dataset were differentially expressed with the same expression pattern in the presence of the 3D scaffold. As expected, 480 common DEGs found in both 2D and 3D scaffold data sets were involved in nervous system related biological processes and functions, like neuron differentiation (FDR q-val = 0.0046) (Fig. 1e and 1f, Table S3). Despite similarities, the expression of many genes was different between 2D and 3D scaffolds, with many more upregulated genes in the 3D culturing condition. Because tissue cells grow in a 3D structure, we hypothesized that neuronal cells differentiated on the 3D scaffold would be more similar to their in vivo counterparts. Therefore, we expected that these up-regulated genes in the 3D culturing condition would be associated with nervous system development. We performed ontology analysis for the most upregulated genes that were exclusive to the 2D biomaterial and we found that the most affected terms were related to nervous system development and functions, including synapse assembly, chemical synaptic transmission, and anterograde trans-synaptic signaling (Fig. 1g). We performed the same analysis for the most upregulated genes exclusive to the 3D biomaterial, and unexpectedly found a high number of terms associated with inflammatory response (Fig. 1h). The 2D most upregulated terms lacked any that were related to immune function. Overall, the transcriptomic profile of neural progenitor cells cultured in 2D and 3D scaffolds revealed that both induce common gene expression related to nervous system development. In addition to these genes, however, the 3D scaffold induces more biological processes that are not related to nervous system development, including inflammatory responses. Therefore, in contrast to our hypothesis, it seems culturing of NPCs on 2D porous polystyrene produce neurons with more mature cell characteristics according to their transcriptome profile, however, further experimental studies are needed to confirm our result in more detail.
Dimensionality is an important contributor to stem cell differentiation and relatedness to in vivo counterparts. However, there are other modalities of biomaterials that can influence the success of differentiation. Here, we investigate the role of degradable vs non-degradable biomaterials as one influencer. Gjorevski et al. used either degradable or non-degradable enzymatically crosslinked polyethylene glycol (PEG) during epithelial organoid formation from intestinal stem cells (ISCs) (Gjorevski et al., 2016). Remodeling is important for repair and morphogenesis, so they hypothesized that the degradable matrix would improve organoid formation. Interestingly, their results indicated that the use of degradable PEG resulted in an abnormal shape of colonies and fewer organoids formed. The transcriptomic profile of ISCs demonstrated that genes related to stress and inflammation were significantly enriched in ISCs cultured in the presence of degradable PEG compared to non-degradable PEG. We re-analyzed their RNA-seq data to further investigate the possible mechanism of the poor organoid formation from ISCs cultured in the degradable PEG.
Comparing ISC differentiation in the presence of degradable and non-degradable PEGs showed 170 DEGs (FC>2 andp-value <0.05). Among these, 115 genes were upregulated in degradable PEGs, and the majority were associated with an inflammatory response and a positive regulation of leukocyte chemotaxis in comparison with non-degradable PEGs (Fig. 2a). To find the main regulators of the inflammatory response related genes in ISCs differentiation, we generated a gene regulatory network containing 33 genes (nodes) and 53 interactions between them (edges) (Fig. 2b). Analysis of this constructed gene regulatory network revealed ATF3 and KLF6 as the main regulators of the inflammatory response (Fig. 2c). In addition, we constructed another network using a list of differentially expressed TFs, called protein-protein interaction network (Fig. 2d). In the constructed protein-protein interaction network, ATF3 and KLF6 are found to interact with NFKBIA that subsequently interacts with TNF. Interestingly, it was previously shown that KLF6 expression dramatically increased in both intestinal tissue and myeloid cells in inflammatory bowel disease (Goodman et al., 2016). So, it seems that recruitment of transcription factors ATF3 and KLF6 could be a possible mechanism for activation inflammatory cascaded in abnormal organoid formation.