Well-organized spheroids as a new platform to examine cell interaction and behaviour during organ development

Abstract

Single-cell analysis provides molecular signatures to define cell identity. To characterize cell types, DNA methylation patterns are often used as a flag of internal molecular status. There are a few reports of single-cell methylome techniques that involves bisulfite conversion. However, the step often causes DNA fragmentation, which leads to severe PCR substrate reduction. Here we developed a new version of single-cell reduced representation bisulfite sequencing (scRRBS) method to recover more CpG sites to be analysed. Our method succeeded to increase of 4.1 times in sample yield and 1.6 times in CpG site coverage. Importantly, our results indicate that the obtained single-cell DNA methylation sites are homogeneous among 12 cells, thus it may provide molecular barcodes for cell types. In summary, we succeed to develop enhanced scRRBS method and it will be more useful tool to define cell identity in the near future.

Porphyromonas gingivalis bypasses epithelial barrier and modulates fibroblastic inflammatory response in an in vitro 3D spheroid model

“…It has already been described that the synthesis of 3D MT using hanging-drop method is a reliable technique to generate homogenous organs or tissue-like structures at low-cost without requiring specific growth factors or complicated technical procedures. This type of spheroid model has been used previously to determine the physiopathological processes associated with diseases such as cancers, cellular organization during embryogenesis, drug testing and also as potential therapeutics such as demonstrated in the context of dental pulp regeneration 10 , 17 , 34 – 36 . The use of this model is of interest to evaluate the effects associated with the infection by P. gingivalis or by other periodontal pathogens while taking into account the epithelio-fibroblastic interactions.…”

“…MT is characterized by a 3D spheroid structure where cells are in direct contact, allowing cell-to-cell and cells-to-extracellular matrix components (ECM) interactions. This method can be used to co-culture two (or more) different cell populations to elucidate the role of cell-cell or cell-ECM interactions in a 3D environment 10 , 12 (Fig. 1B ).…”

“…Therefore, to be able to consider the complexity of tissues or organs, 3D cell models have been developed 8 – 11 . 3D models have been described as mimicking more closely the natural tissues and organs physiology and phenotypes than cells grown in 2D 10 , 12 . The spatial proximity of cells in such a model also enables interactions between adhesion molecules and receptors maximizing cellular communication and signaling that is critical to cell function 13 .…”

Bone Marrow Stromal Cells Promote Innervation of Bioengineered Teeth

“…Analysis of messenger RNA (mRNA) expression was investigated as previously documented (Bécavin et al 2016; for further details, see Appendix Method 4 and Appendix Table 2).…”

Development of Enhanced Reduced Representation Bisulfite Sequencing Method for Single-cell Methylome Analysis

“…It is important to know how these organized positioning of cells is configured. Recently some papers reported on cellular interaction events using an organoid, which mimics tissue formation [1].…”