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. 2021 Jun 22;22(13):6663.
doi: 10.3390/ijms22136663.

Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts

Maurycy Jankowski  1 , Mariusz Kaczmarek  2   3 , Grzegorz Wąsiatycz  4 , Claudia Dompe  5 , Paul Mozdziak  6 , Jędrzej M Jaśkowski  7 , Hanna Piotrowska-Kempisty  8   9 , Bartosz Kempisty  1   4   6   10
Affiliations

Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts

Maurycy Jankowski et al. Int J Mol Sci. .

Abstract

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic differentiation of ASCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture and osteogenic differentiation of ASCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ASCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells' application in regenerative medicine.

Keywords: RNAseq; adipose; analysis; stem cells; transcriptome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The results of flow cytometry analysis of selected ASC markers in the cell samples subjected to in vitro culture.
Figure 2
Figure 2
The results of morphological analysis of cASC primary culture (monochrome), combined with Alizarin Red staining photographs (color), to confirm osteogenic differentiation of the cells of interest. Scale bar: 100 μm.
Figure 3
Figure 3
Principal component analysis of cASC samples subjected to RNAseq analysis.
Figure 4
Figure 4
Volcano plots representing the composition of the analyzed sample groups, as well as the proportion and distribution of differentially expressed genes. (A)—day 14 vs. day 1, (B)—differentiated osteoblast vs. day 1, (C)—differentiated osteoblast vs. day 14.
Figure 5
Figure 5
The heatmap representing the changes in the 10 most up- and downregulated genes between day 1 and day 14 of primary cASC culture, presented as log2FC.
Figure 6
Figure 6
The heatmap representing the changes in the 10 most up- and downregulated genes between day 1 of primary cASC culture and differentiated osteoblasts, presented as log2FC.
Figure 7
Figure 7
The heatmap representing the changes in the 10 most up- and downregulated genes between day 14 of primary cASC culture and differentiated osteoblasts, presented as log2FC.
Figure 8
Figure 8
Results of the STRING analysis of predicted interactions between protein products encoded by the differentially analyzed genes of interest. The genes not involved in any interactions were excluded from the figure. (A)—day 14 vs. day 1, (B)—differentiated osteoblast vs. day 1, (C)—differentiated osteoblast vs. day 14. Colors of the edges indicate the source of predicted interaction: magenta—experimentally determined, green—textmining, black—co-expression.
Figure 9
Figure 9
The results of RT-qPCR validation analysis presented as log2FC.
See this image and copyright information in PMC

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