Andrographolide promotes proliferative and osteogenic potentials of human placenta-derived mesenchymal stem cells through the activation of Wnt/β-catenin signaling

Abstract

Introduction The in vitro expansion and differentiation of mesenchymal stem cells derived from bone marrow (BM-hMSCs) are considered as potential therapeutic tools for clinical applications in bone tissue engineering and regenerative medicine. However, invasive sampling and reduction in number and proliferative capacity with age are the major limitations of BM-hMSCs. Recently, human placenta-derived MSCs (PL-hMSCs) obtained by a non-invasive procedure have attracted much interest. Attempts to increase the potential of PL-hMSCs would be an important paradigm in regenerative medicine. Herein, we examined the proliferative and osteogenic effect of andrographolide (AP) on PL-hMSCs. Methods Mesenchymal stem cells were isolated from full-term normal human placentas and were characterized before using. Cell cytotoxicity and proliferative effect of AP were examined by MTT and BrdU assay, respectively. The non-toxicity concentrations of AP were further assessed for osteogenic effect determined by alkaline phosphatase (ALP) expression and activity, alizarin red staining, and osteoblast-specific gene expressions. Screening of genes involved in osteogenic differentiation-related pathways modulated by AP was explored by a NanoString nCounter analysis. Results PL-hMSCs generated in this study met the MSC criteria set by the International Society of Cellular Therapy. The non-cytotoxic concentrations of AP on PL-hMSCs are up to 10 μM. The compound increased PL-hMSC proliferation concomitant with increases in Wnt/β-catenin level and activity. It also enhanced osteogenic differentiation in association with osteoblast-specific mRNA expression. Further, AP promoted bone formation and increased bone structural protein level, osteocalcin, in osteoblastic cells. Gene screening analysis showed the upregulation of genes related to Wnt/β-catenin, TGFβ/BMP, SMAD, and FGF signaling pathways. Conclusion We demonstrated, for the first time, the potential role of AP in promoting proliferation, osteogenic differentiation, and osteoblast bone formation of PL-hMSCs. This study suggests that AP may be an effective novel agent for the improvement of PL-hMSCs and stem cell-based therapy for bone regeneration.

Andrographolide Reduces Lipid Droplet Accumulation in Adipocytes Derived from Human Bone Marrow Mesenchymal Stem Cells by Suppressing Regulators of Adipogenesis

“…Activation of Wnt/β-catenin promotes osteogenesis while suppressing adipogenesis as it enhances the expression of transcription factor RUNX2 and reduces the expression of PPARγ. , On the other hand, Wnt is inhibited by several molecules such as DKK1 and WNT5B, resulting in the activation of PPARγ to enhance the adipogenic differentiation of MSCs . In a previous study, AP has been shown to promote the osteogenic differentiation lineage of MSCs by activating the Wnt signaling pathway and increasing RUNX2 expression . This is consistent with our study in which AP showed an inhibitory effect on the gene expression of DKK1 , WNT5B , and PPARγ .…”

“…The expression of specific surface markers of hBM-MSCs was evaluated by flow cytometry (FACScalibur, Becton Dickinson, USA), as previously described . Briefly, after trypsinization, cells were suspended in PBS and incubated for 30 min at 4 °C in the dark with antihuman monoclonal antibodies including PE-conjugated anti-CD34 antibody, PE-conjugated anti-CD73 antibody, PE-conjugated anti-CD90 antibody, PE-conjugated anti-CD105 antibody, and FITC-conjugated anti-CD45 antibody (Bio Legend, USA).…”

“…The modulatory effect of AP on adipogenic regulatory genes during adipocyte differentiation was assessed using NanoString nCounter technology (NanoString Technologies, Seattle, WA, USA). nCounter PanCancer Panel containing 770 genes was used, and all samples were prepared according to the manufacturer’s instructions as described in our previous study . The target genes were normalized by the geometric mean of 29 housekeeping genes using the nSolver software, version 4.0 analysis.…”

Phoenix dactilyfera L. Pits Extract Restored Bone Homeostasis in Glucocorticoid-Induced Osteoporotic Animal Model through the Antioxidant Effect and Wnt5a Non-Canonical Signaling

“…Therefore, osteoblast's differentiation markers were evaluated by measuring Runx2, Osx, OPG, COL1A1 and OCN. Runx2 and Osx are transcription factors that act downstream of the Wnt/βcatenin signaling in transcribing the bone-forming genes OPG, COL1A1, and OCN [74,75]. DEXA injections revealed a significant decrease in the expression levels of bone Runx2 and Osx and their downstream targets (COL1A1, OCN, and OPG), indicating a deactivated osteoblasts Wnt/β-catenin signaling.…”

PPARγ-Induced Global H3K27 Acetylation Maintains Osteo/Cementogenic Abilities of Periodontal Ligament Fibroblasts

“…The secretion of Wnt proteins, such as Wnt3a and Wnt5a, which are inducers of osteo/cementoblastic differentiation [16,17], was eliminated in the Osteocalcin-expressing cells of conditional knockout mice of Wntless [15]. Osteocalcin was shown to be specifically expressed in cells differentiating towards or differentiated osteo/cementoblastic lineage cells, but was negligible in stem cells [18]; however, it was expressed in PDLF [19]. In the periodontal tissue of conditional knockout mice of Wntless, the periodontal ligament and cementum were wider, the alveolar bone was narrower, and the expression of osteogenic markers, such as Alp, Runx2, and Osterix, was downregulated [15].…”