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. 2022 Aug 28:2022:2116367.
doi: 10.1155/2022/2116367. eCollection 2022.

Collagen Modulates the Biological Characteristics of WJ-MSCs in Basal and Osteoinduced Conditions

Vun Vun Hiew  1 , Peik Lin Teoh  1
Affiliations

Collagen Modulates the Biological Characteristics of WJ-MSCs in Basal and Osteoinduced Conditions

Vun Vun Hiew et al. Stem Cells Int. .

Abstract

Transcriptomic analysis revealed mesenchymal stem/stromal cells (MSCs) from various origins exhibited distinct gene and protein expression profiles dictating their biological properties. Although collagen type 1 (COL) has been widely studied in bone marrow MSCs, its role in regulating cell fate of Wharton jelly- (WJ-) MSCs is not well understood. In this study, we investigated the effects of collagen on the characteristics of WJ-MSCs associated with proliferation, surface markers, adhesion, migration, self-renewal, and differentiation capabilities through gene expression studies. The isolated WJ-MSCs expressed positive surface markers but not negative markers. Gene expression profiles showed that COL not only maintained the pluripotency, self-renewal, and immunophenotype of WJ-MSCs but also primed cells toward lineage differentiations by upregulating BMP2 and TGFB1 genes. Upon osteoinduction, WJ-MSC-COL underwent osteogenesis by switching on the transcription of BMP6/7 and TGFB3 followed by activation of downstream target genes such as INS, IGF1, RUNX2, and VEGFR2 through p38 signalling. This molecular event was also accompanied by hypomethylation at the OCT4 promoter and increase of H3K9 acetylation. In conclusion, COL provides a conducive cellular environment in priming WJ-MSCs that undergo a lineage specification upon receiving an appropriate signal from extrinsic factor. These findings would contribute to better control of fate determination of MSCs for therapeutic applications related to bone disease.

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Conflict of interest statement

The authors declare that there is no conflict of interest regarding the publication of this paper.

Figures

Figure 1
Figure 1
Morphological observation and proliferation of WJ-MSCs. (a) P0, (b) P1, (c) P3, (d) P6, (e) P9, and (f) P11. Scale bar = 200 μm. (g) Cells at P3, P6, and P9 were cultured for up to 20 days. Data obtained from three biological independent samples.
Figure 2
Figure 2
The mRNA and protein expression of surface markers in WJ-MSC. (a) The gene expression of positive surface markers was quantified after relative to ACTB. Data obtained from three independent biological samples. Asterisk (∗) indicates statistical significance (p < 0.5). (b) Immunocytochemical staining was performed against CD90 (green), CD14 (red), and CD34 (red). The cells were counter-stained using DAPI (blue).
Figure 3
Figure 3
Trilineage differentiation potentials of WJ-MSCs. Cells cultured in basal and induction media were stained with (a) Alizarin red, (b) Oil red O, and (c) Alcian blue.
Figure 4
Figure 4
The effects of collagen on the morphology, proliferation, and lineage differentiation of WJ-MSCs. (a) Cell morphological observation at P3 and P6 on a collagen-coated plate. (b) Relative proliferation of cells cultured on collagen plate compared to the control. Data obtained from three independent biological samples. (c) Staining of differentiated cells cultured on the collagen surface with or without induction.
Figure 5
Figure 5
The effects of collagen on the gene expression of surface markers. WJ-MSC-COL cultured in basal media was compared to WJ-MSCs without COL, while WJ-MSC-COL cultured in osteogenic media was compared to WJ-MSC-COL in basal media. All data were obtained from three independent biological samples. Asterisk (∗) indicates statistical significance (p < 0.05).
Figure 6
Figure 6
Collagen modulated the expression of differentiation genes. The expression of (a) osteogenic, (b) chondrogenic, (c) adipogenic, and (d) other lineage differentiation genes. WJ-MSC-COL cultured in basal media was compared to WJ-MSCs without COL, while WJ-MSC-COL cultured in osteogenic media was compared to WJ-MSC-COL in basal media. Asterisk (∗) indicates statistical significance (p < 0.05). The CEBPβ expression was obtained through semiquantitative RT-PCR. All data were obtained from three independent biological samples.
Figure 7
Figure 7
Collagen modulated the expression of (a) stemness, (b) immunomodulatory, and (c) other MSC genes. WJ-MSC-COL cultured in basal media was compared to WJ-MSCs without COL, while WJ-MSC-COL cultured in osteogenic media was compared to WJ-MSC-COL in basal media. Asterisk (∗) indicates statistical significance (p < 0.05). The SOX2 and NANOG expressions were obtained through semiquantitative RT-PCR. All data obtained from three independent biological samples.
Figure 8
Figure 8
Collagen influenced the gene expression of epigenetic regulators. Cells were cultured on collagen scaffold at basal or osteogenic condition. (a) The gene expression of histone acetyltransferases. (b) Representative Western blot was probed with antibodies as indicated. Relative protein expression level obtained after normalization using histone H3. (c) Top: the gene expression of DNA methyltransferases; bottom: the methylation status at the promoter regions of OCT4 and SOX2. Circles represent the CpG sites. All data were obtained from three independent biological samples. Asterisk (∗) indicates significance (p < 0.05).
Figure 9
Figure 9
MAPK pathways mediated osteogenesis in WJ-MSCs. Cells were cultured on collagen scaffold at basal or osteogenic condition. (a) Representative Western blot was probed with antibodies as indicated. (b) Relative protein expression level obtained after normalization using histone H3. Data were obtained from three independent biological samples. Asterisk (∗) indicates statistical significance (p < 0.05).
Figure 10
Figure 10
Summary of the underlying mechanisms of collagen modulating lineage differentiation in WJ-MSCs under basal and induced conditions.
See this image and copyright information in PMC

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