Int J Stem Cells.  2024 Feb;17(1):91-98. 10.15283/ijsc23043.

Mimicking the Human Articular Joint with In Vitro Model of Neurons-Synoviocytes Co-Culture

• Chwastek J
• Kędziora M
• Borczyk M
• Korostyński M
• Starowicz K

• KMID: 2552023
• DOI: http://doi.org/10.15283/ijsc23043

Abstract

The development of In Vitro models is essential in modern science due to the need for experiments using human material and the reduction in the number of laboratory animals. The complexity of the interactions that occur in living organisms requires improvements in the monolayer cultures. In the work presented here, neuroepithelial stem (NES) cells were differentiated into peripheral-like neurons (PLN) and the phenotype of the cells was confirmed at the genetic and protein levels. Then RNA-seq method was used to investigate how stimulation with pro-inflammatory factors such as LPS and IFNγ affects the expression of genes involved in the immune response in human fibroblast-like synoviocytes (HFLS). HFLS were then cultured on semi-permeable membrane inserts, and after 24 hours of pro-inflammatory stimulation, the levels of cytokines secretion into the medium were checked. Inserts with stimulated HFLS were introduced into the PLN culture, and by measuring secreted ATP, an increase in cell activity was found in the system. The method used mimics the condition that occurs in the joint during inflammation, as observed in the development of diseases such as rheumatoid arthritis (RA) or osteoarthritis (OA). In addition, the system used can be easily modified to simulate the interaction of peripheral neurons with other cell types.

Keyword

Neuroepithelial cells; Synoviocytes; Inflammation; Joint disorders

Figure

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  Fig. 1 Confirmation of cell differentiation by qPCR, measuring marker genes (NTRK1 [A] and PRPH [B]) for peripheral neurons. Results from peripheral-like neurons (PLN) were normalized to the reference gene (B2M) and expression level in undifferentiated cells, and results are presented as mean±SEM (n=6 or 8 for control and test groups, respectively). Data were analyzed with Student’s t-test with ***p<0.001. Immunocytochemical staining was done to demonstrate expression of marker proteins (PRPH and MAP2) and cell nuclei (DAPI) in day 2 (C) and 28 (D) of culture.

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  Fig. 2 Human fibroblast-like synoviocytes (HFLS) secretome analysis after pro-inflammatory stimulation. Pro-inflammatory compounds activated HFLS and led to upregulation of genes encoding proteins involved in immune response. For the heatmap example top 41 differentially regulated genes (DEGs) from RNA-seq data were selected based on the following criteria: false discovery rate (FDR)<0.01, at least one post-hoc t-test with p-value<1e-8, and absolute value of log2FC>3.5 (A). Lipopolysaccharide (LPS) stimulation is more effective than IFNγ as shown in the Venn diagram displaying numbers of DEGs by stimulant (FDR<0.01, post-hoc t-test p-value<0.01) (B), but both factors significantly modulated immunological processes (Gene Ontology [GO] term enrichment was performed on lists of significantly upregulated DEGs) (C). Pro-inflammatory stimulated HFLS released to media IL6 (D) and CCL2 (E), which migrated through semipermeable membrane. ELISA results are presented as mean±SEM (n=4) and analyzed by one-way ANOVA followed by Tukey’s post hoc test with *p<0.05 and **p<0.01.

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  Fig. 3 Pro-inflammatory stimulated peripheral-like neurons (PLN) secreting adenozyno-5’-trifosforan (ATP) into media. As shown in the diagram (A), PLN were stimulated by 10 ng/ml pro-inflammatory factors (LPS or IFNγ) (B, C) or by co-culture with immunologically active human fibroblast-like synoviocytes (HFLS) (previously stimulated with LPS or IFNγ) (D, E). Data were calculated as % control (unstimulated PLN or co-culture with unstimulated HFLS for each time point) and presented as mean±SEM (n=4). Results were analyzed by one-way ANOVA followed by Dunnet’s post hoc test with *p<0.05, **p<0.01, and ***p<0.001.

Reference

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