Multi-lineage differentiation of mesenchymal stem cells - To Wnt, or not Wnt

Mesenchymal stem cells (MSCs) are multipotent precursor cells originating from several adult connective tissues. MSCs possess the ability to self-renew and differentiate into several lineages, and are recognized by the expression of unique cell surface markers. Several lines of evidence suggest that various signal transduction pathways and their interplay regulate MSC differentiation. To that end, a critical player in regulating MSC differentiation is a group of proteins encoded by the Wnt gene family, which was previously known for influencing various stages of embryonic development and cell fate determination. As MSCs have gained significant clinical attention for their potential applications in regenerative medicine, it is imperative to unravel the mechanisms by which molecular regulators control differentiation of MSCs for designing cell-based therapeutics. It is rather coincidental that the functional outcome(s) of Wnt-induced signals share similarities with cellular redox-mediated networks from the standpoint of MSC biology. Furthermore, there is evidence for a crosstalk between Wnt and redox signalling, which begs the question whether Wnt-mediated differentiation signals involve the intermediary role of reactive oxygen species. In this review, we summarize the impact of Wnt signalling on multi-lineage differentiation of MSCs, and attempt to unravel the intricate interplay between Wnt and redox signals

Wnt antagonist secreted frizzled-related protein 4 upregulates adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells

With more than 1.4 billion overweight or obese adults worldwide, obesity and progression of the metabolic syndrome are major health and economic challenges. To address mechanisms of obesity, adipose tissue-derived mesenchymal stem cells (ADSCs) are being studied to detail the molecular mechanisms involved in adipogenic differentiation. Activation of the Wnt signalling pathway has inhibited adipogenesis from precursor cells. In our study, we examined this anti-adipogenic effect in further detail stimulating Wnt with lithium chloride (LiCl) and 6-bromo indirubin 3'oxime (BIO). We also examined the effect of Wnt inhibition using secreted frizzled-related protein 4 (sFRP4), which we have previously shown to be pro-apoptotic, anti-angiogenic, and anti-tumorigenic. Wnt stimulation in LiCl and BIOtreated ADSCs resulted in a significant reduction (2.7-fold and 12-fold respectively) in lipid accumulation as measured by Oil red O staining while Wnt inhibition with sFRP4 induced a 1.5-fold increase in lipid accumulation. Furthermore, there was significant 1.2-fold increase in peroxisome proliferator-activated receptor gamma (PPAR ?) and CCAAT/enhancer binding protein alpha (C/EBPa), and 1.3-fold increase in acetyl CoA carboxylase protein levels. In contrast, the expression of adipogenic proteins (PPAR?, C/EBPa, and acetyl CoA carboxylase) were decreased significantly with LiCl (by 1.6, 2.6, and 1.9-fold respectively) and BIO (by 7, 17, and 5.6-fold respectively) treatments. These investigations demonstrate interplay between Wnt antagonism and Wnt activation during adipogenesis and indicate pathways for therapeutic intervention to control this process

Implications of the WNT Signalling Pathway for Adipose-derived Mesenchymal Stem Cells in a Breast Tumour Environment

This study describes the various roles adipose-derived mesenchymal stem cells (ADSCs) play in a breast tumour environment. The ADSC-derived secreted factors inhibit breast tumour cell growth aspects, whereas the tumour-derived secreted factors transform ADSCs into tumour-associated fibroblasts (TAFs). Additionally, it highlights the role of Wnt antagonist sFRP4 and its peptides to further influence the activity of ADSC-derived secreted factors, to downregulate the transformation of ADSCs into TAFs, and in upregulating the adipogenic differentiation of ADSCs

The role of Wnt signalling in angiogenesis

Angiogenesis is a normal biological process wherein new blood vessels form from the growth of pre-existing blood vessels. Preventing angiogenesis in solid tumours by targeting pro-angiogenic factors including vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), basic fibroblast growth factor (bFGF), hepatocyte growth factor, and platelet-derived growth factor (PDGF) is currently under investigation for cancer treatment. Concurrently targeting the cell signalling pathways involved in the transcriptional and post-translational regulation of t hese factors may provide positive therapeutic results. One such pathway is the Wnt signalling pathway. Wnt was first discovered in mice infected with mouse mammary tumour virus, and has been crucial in improving our understanding of oncogenesis and development. In this review, we summarise molecular and cellular aspects of the importance of Wnt signalling to angiogenesis, including ß-catenin-dependent mechanisms of angiogenic promotion, as well as the study of Wnt antagonists, such as the secreted frizzled-related protein family (SFRPs) which have been shown to inhibit angiogenesis. The growing understanding of the underlying complexity of the biochemical pathways mediating angiogenesis is critical to the identification of new molecular targets for therapeutic applications

Dose response of ADSCs with Wnt regulators.

<p>In the presence of Wnt activators (A) LiCl, (B) BIO, and (C) Wnt antagonist sFRP4 (* p<0.05 and ** p<0.001).<b>iiiiiiivviv</b></p

Differentiation of ADSCs visualised by staining techniques.

<p>(A) Intracellular lipid globules of adipogenically differentiated ADSCs stained positively by Oil Red O (B) Calcium deposition of osteogenically differentiated ADSCs stained positively by Alizarin Red, (C) Mineralization of osteogenically differentiated ADSCs stained positively by Von Kossa, (D) Glycosaminoglycan deposition of chondrogenically differentiated ADSCs stained positively by Alcian blue, and (E) Fibroblast morphology of undifferentiated ADSCs. Scale bar = 10μM.</p

Morphology of ADSCs during adipogenic differentiation.

<p>At (A) Day 1 and (B) Day 7. Treatment conditions were (i) non-induced control media, (ii) adipogenic control, (iii) 10mM LiCl, (iv) 0.5μM BIO, (v) 100pg/mL sFRP4, and (vi) 1ng/mL sFRP4 in both (A) and (B). Scale bar = 10μM.</p

Morphology of ADSCs during adipogenic differentiation at Day 7.

<p>(A) Before and (B) after Oil red O staining. Treatment conditions were (i) non-induced control media, (ii) adipogenic control, (iii) 0.5μM BIO, (iv) 1ng/mL sFRP4, and (v) 0.5μM BIO + 1ng/mL sFRP4 in both (A) and (B). Scale bar = 10μM in (A) and 250μM in (B). (C) Quantification of the stained lipid droplets were performed using the eluted Oil red O stain via measuring absorbance at 510nm. The readings were normalised to background values of non-induced control ADSCs. The values of all treatment conditions were compared to the adipogenic control group (** p<0.001).</p

Oil Red O Staining and quantification on Day 7.

<p>(A) Microscopic observations of the stained lipid droplets. Treatment conditions were (i) non-induced control, (ii) adipogenic control, (iii) 10mM LiCl, (iv) 0.5μM BIO, (v) 100pg/mL sFRP4, and (vi) 1ng/mL sFRP4. Scale bar = 250μM (B) Quantification of the stained lipid droplets were performed using the eluted Oil red O stain via measuring absorbance at 510nm. The readings were normalised to background values of non-induced control ADSCs. The values of all treatment conditions were compared to the adipogenic control group (* p<0.05 and ** p<0.001).</p

The Influence of Breast Tumour-Derived Factors and Wnt Antagonism on the Transformation of Adipose-Derived Mesenchymal Stem Cells into Tumour-Associated Fibroblasts

Within the tumour stroma, a heterogeneous population of cell types reciprocally regulates cell proliferation, which considerably affects the progression of the disease. In this study, using tumour conditioned medium (TCM) derived from breast tumour cell lines – MCF7 and MDA MB 231, we have demonstrated the differentiation of adipose-derived mesenchymal stem cells (ADSCs) into tumour-associated fibroblasts (TAFs). Since the Wnt signalling pathway is a key signalling pathway driving breast tumour growth, the effect of the Wnt antagonist secreted frizzled-related protein 4 (sFRP4) was also examined. The response of ADSCs to TCM and sFRP4 treatments was determined by using cell viability assay to determine the changes in ADSC viability, immunofluorescence for mesenchymal markers, glucose uptake assay, and glycolysis stress test using the Seahorse Extracellular Flux analyser to determine the glycolytic activity of ADSCs. ADSCs have been shown to acquire a hyper-proliferative state, significantly increasing their number upon short-term and long-term exposure to TCM. Changes have also been observed in the expression of key mesenchymal markers as well as in the metabolic state of ADSCs. SFRP4 significantly inhibited the differentiation of ADSCs into TAFs by reducing cell growth as well as mesenchymal marker expression (cell line-dependent). However, sFRP4 did not induce further significant changes to the altered metabolic phenotype of ADSCs following TCM exposure. Altogether, this study suggests that the breast tumour milieu may transform ADSCs into a tumour-supportive phenotype, which can be altered by Wnt antagonism, but is independent of metabolic changes