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. 2023 Oct 26;14(1):305.
doi: 10.1186/s13287-023-03533-y.

Regulation of self-renewal and senescence in primitive mesenchymal stem cells by Wnt and TGFβ signaling

Matteo Mazzella  1   2 , Keegan Walker  1   2 , Christina Cormier  1   2 , Michael Kapanowski  1   2 , Albi Ishmakej  1   2 , Azeem Saifee  1   2 , Yashvardhan Govind  1   2 , G Rasul Chaudhry  3   4
Affiliations

Regulation of self-renewal and senescence in primitive mesenchymal stem cells by Wnt and TGFβ signaling

Matteo Mazzella et al. Stem Cell Res Ther. .

Abstract

Background: The therapeutic application of multipotent mesenchymal stem cells (MSCs) encounters significant challenges, primarily stemming from their inadequate growth and limited self-renewal capabilities. Additionally, as MSCs are propagated, their ability to self-renew declines, and the exact cellular and molecular changes responsible for this are poorly understood. This study aims to uncover the complex molecular mechanisms that govern the self-renewal of primitive (p) MSCs.

Methods: We grew pMSCs using two types of medium, fetal bovine serum (FM) and xeno-free (XM), at both low passage (LP, P3) and high passage (HP, P20). To evaluate LP and HP pMSCs, we examined their physical characteristics, cell surface markers, growth rate, colony-forming ability, BrdU assays for proliferation, telomerase activity, and potential to differentiate into three lineages. Moreover, we conducted RNA-seq to analyze their transcriptome and MNase-seq analysis to investigate nucleosome occupancies.

Results: When grown in FM, pMSCs underwent changes in their cellular morphology, becoming larger and elongated. This was accompanied by a decrease in the expression of CD90 and CD49f, as well as a reduction in CFE, proliferation rate, and telomerase activity. In addition, these cells showed an increased tendency to differentiate into the adipogenic lineage. However, when grown in XM, pMSCs maintained their self-renewal capacity and ability to differentiate into multiple lineages while preserving their fibroblastoid morphology. Transcriptomic analysis showed an upregulation of genes associated with self-renewal, cell cycle regulation, and DNA replication in XM-cultured pMSCs, while senescence-related genes were upregulated in FM-cultured cells. Further analysis demonstrated differential nucleosomal occupancies in self-renewal and senescence-related genes for pMSCs grown in XM and FM, respectively. These findings were confirmed by qRT-PCR analysis, which revealed alterations in the expression of genes related to self-renewal, cell cycle regulation, DNA replication, differentiation, and senescence. To understand the underlying mechanisms, we investigated the involvement of Wnt and TGFβ signaling pathways by modulating them with agonists and antagonists. This experimental manipulation led to the upregulation and downregulation of self-renewal genes in pMSCs, providing further insights into the signaling pathways governing the self-renewal and senescence of pMSCs.

Conclusion: Our study shows that the self-renewal potential of pMSCs is associated with the Wnt pathway, while senescence is linked to TGFβ.

Keywords: Differentiation; Mesenchymal stem cells; Proliferation; Self-renewal; Senescence; TGFβ signaling; Wnt pathway.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Characteristics of LP and HP pMSCs grown in FM and XM. a, b Phase contrast microscopy images and expression of surface markers determined by flow cytometer in pMSCs, respectively. Scale bars represent 100 μm (magnification: 4×). HP pMSCs in FM had larger cell size and significantly downregulated expression of CD49f and CD90 compared to LP pMSCs
Fig. 2
Fig. 2
Proliferation and colony-forming efficiency assays of LP and HP pMSCs grown in FM and XM. a Doubling time of pMSCs b, c Crystal violet stained colonies of pMSCs and phase contrast showing the morphology of a single colony of pMSCs performed in triplicate. Scale bars represent 100 μm (magnification: 4×). d Percentage of colony formation of LP and HP pMSCs performed in triplicate. E Proliferation of pMSCs as determined using BrdU proliferation kit performed in triplicate. f Relative telomerase activity in various cell types as determined using the qRT-PCR-based TRAP assay performed in triplicate. All values are reported as telomerase activity relative to HEK cells. Growth of pMSCs in FM yielded cells with significantly reduced doubling time and CFE, less compact colonies, and decreased proliferation rate and relative telomerase activity. g Light microscopy images of SA-β-gal stained pMSCs. Blue staining indicates senescent cells. Scale bars represent 100 μm (magnification: 4×). Any results showing **p ≤ 0.01 and *p ≤ 0.05 were deemed statistically significant
Fig. 3
Fig. 3
Determination of differentiation potential of LP and HP pMSCs grown in FM and XM. a–c Fluorescence microscopic images and fluorescence intensity of the derivatives stained with CEBP, COL2, and OCN antibodies representing chondrogenic, osteogenic, and adipogenic lineages, respectively. d Quantification of fluorescent intensity of immunostained derivatives of pMSCs performed in triplicate. Scale bars represent 400 μm (magnification: 40×). e Expression of adipogenic (CEBPβ, FABP4, and PPARγ), chondrogenic (SOX9, ACAN, and COL2), and osteogenic (COL1, RUNX2, OPN, and OCN) genes in the differentiated derivatives. Gene expression was normalized to GAPDH and ACTIN, and error bars represent the standard deviations of the triplicate measurements. HP pMSCs grown in FM showed a greater propensity to differentiate toward adipogenic lineage. Any results showing **p ≤ 0.01 and *p ≤ 0.05 were deemed statistically significant
Fig. 4
Fig. 4
Transcriptional analysis in LP and HP pMSCs grown in FM and XM. a–e RNA-seq results show the Wnt signaling, cell cycle, DNA replication, TGF beta, and senescence genes, respectively, performed in triplicate. Up- and downregulated genes are red and blue, respectively. HP pMSCs grown in FM displayed downregulation of Wnt signaling, cell cycle, and DNA replication genes but upregulation of TGF beta and senescence genes. f–h Enrichment of upregulated DEGs associated with the biological processes, molecular function, and cellular compartment, respectively, was performed with Benjamini–Hochberg FDR at p < 0.05 using DESeq2. i–j Upregulated DEGs associated with protein classes and signaling pathways, respectively, were determined by PANTHER analysis. k Enrichment of pathway genes when DEGs were compared between HP XM vs HP FM and LP XM vs LP FM. The Enrichr analysis generated the combined score (p-value multiplied by the z-score)
Fig. 5
Fig. 5
Nucleosome mapping and genome browser shots showing nucleosome occupancy determined by Mnase-seq and IGV analysis a–c Nucleosome mapping showing cell cycle, DNA replication, and senescence genes, respectively. d–f Wnt pathway genes (FZD1, LRP6, and Wnt2B), g–j VEGF/PDGF pathway genes (VEGFA, FLT1, PDGFC, and PDGFRA), k cell cycle gene (PSMD7), L–M Self-renewal and DNA replication genes (MYC and PCNA), and n–o senescence (CXCL8 and CDKN2A). Data showed less nucleosome occupancies in pMSCs cultured in XM compared to FM in cell cycle and DNA replication genes. In contrast, more nucleosome occupancies in pMSCs cultured in XM than FM were found in senescence genes. IGV mapping showed that FZD1, LRP6, Wnt2B, VEGFA, FLT1, PDGFC, PDGFRA, PSMD7, MYC, and PCNA genes had less nucleosome occupancies around the promoters, suggesting the higher expression in pMSCs grown in XM than FM, whereas CXCL8 and CDKN2A had higher nucleosome occupancies around the promoters, suggesting their lower expression in pMSCs grown in XM compared to FM. LP and HP pMSCs grown in FM (blue and green, respectively), LP and HP pMSCs grown in XM (orange and purple, respectively)
Fig. 6
Fig. 6
Relative expression of genes in pMSCs cultured in FM and XM. a Genes involved in self-renewal, cell cycle, and DNA replication. b Genes involved in differentiation and senescence. Gene expression was normalized to GAPDH and ACTIN, and error bars represent the standard deviations of the triplicate measurements. Any results showing **p ≤ 0.01 and *p ≤ 0.05 were deemed statistically significant
Fig. 7
Fig. 7
Effect of Wnt and TGFβ agonist and antagonist on pMSCs cultured in FM and XM. a, b The proliferation of pMSCs cultured in the 10 mM Wnt agonist, LiCl, and 500 nM Wnt antagonist, TFA, determined by direct counts and using BrdU proliferation assay. c Relative expression of selected genes involved in self-renewal in pMSCs cultured in media containing LiCl or TFA compared to the control. d, e The proliferation of cells cultured in 0.25 mM TGFβ agonist, BMPSB4, and 0.125 mM TGFβ antagonist, Asiaticoside, determined by direct counts and using BrdU proliferation assay. f Relative expression of selected genes involved in TGFβ and self-renewal in cells supplemented with BMPSB4 and Asiaticoside compared to the control Gene expression was normalized to GAPDH and ACTIN, and error bars represent the standard deviations of the triplicate measurements. Significance was measured in comparison of the control. Any results showing **p ≤ 0.01 and *p ≤ 0.05 were deemed statistically significant
Fig. 8
Fig. 8
Proposed Wnt, VEGF/PDGF, and TGF signaling pathways mediating self-renewal and cell cycle arrest in pMSCs. In XM, the Wnt protein binds to the Frizzled receptor, keeping the Wnt pathway active. This destabilizes the destruction complex (DVL/AXIN/APC/GSK3β/CK1) and allows β-catenin to promote the expression of self-renewal genes in the nucleus. Additionally, growth factors like VEGF and PDGF play a role in pMSC proliferation in XM by phosphorylating their respective receptors. This recruits the adaptor protein GRB2 and the nucleotide exchange factor SOS, which activate RAS, RAF, MEK, and ERK in sequence. Phosphorylated ERK enters the nucleus and activates the transcription of cell proliferation genes such as c-MYC. Alternatively, ERK activates RSK, which then activates proliferation genes. In FM, PI3K is activated, leading to an increase in PTEN and PDK1 expression, which activates AKT. Activated AKT presumably phosphorylates MDM2, modulating p53 activity responsible for senescence gene expression. Senescence is gradually induced by TGFβ interaction with TGFβR, which phosphorylates SMAD2/3, moving to the nucleus with SMAD4 to turn on genes involved in cell cycle arrest
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