TRPC expression in mesenchymal stem cells

Transient receptor potential canonical (TRPC) channels are key players in calcium homeostasis and various regulatory processes in cell biology. Little is currently known about the TRPC subfamily members in mesenchymal stem cells (MSC), where they could play a role in cell proliferation. We report on the presence of TRPC1, 2, 4 and 6 mRNAs in MSC. Western blot and immunofluorescence staining indicate a membrane and intracellular distribution of TRPC1. Furthermore, the decrease in the level of TRPC1 protein caused by RNA interference is accompanied by the downregulation of cell proliferation. These results indicate that MSC express TRPC1, 2, 4 and 6 mRNA and that TRPC1 may play a role in stem cell proliferation.     

Wnt and Frizzled RNA expression in human mesenchymal and embryonic (H7) stem cells

Background

Wnt signals are important for embryonic stem cells renewal, growth and differentiation. Although 19 Wnt, 10 Frizzled genes have been identified in mammals, their expression patterns in stem cells were largely unknown.

Results

We conducted RNA expression profiling for the Wnt ligands, their cellular receptors "Frizzleds" and co-receptors LRP5/6 in human embryonic stem cells (H7), human bone marrow mesenchymal cells, as well as mouse totipotent F9 teratocarcinoma embryonal cells. Except failing to express Wnt2 gene, totipotent F9 cells expressed RNA for all other 18 Wnt genes as well as all 10 members of Frizzled gene family. H7 cells expressed RNA for each of the 19 Wnt genes. In contrast, human mesenchymal cells did not display detectable RNA expression of Wnt1, Wnt8a, Wnt8b, Wnt9b, Wnt10a, and Wnt11. Analysis of Frizzled RNAs in H7 and human mesechymal cells revealed expression of 9 members of the receptor gene family, except Frizzled8. Expression of the Frizzled co-receptor LRP5 and LRP6 genes were detected in all three cell lines. Human H7 and mouse F9 cells express nearly a full complement of both Wnts and Frizzleds genes. The human mesenchymal cells, in contrast, have lost the expression of six Wnt ligands, i.e. Wnt1, 8a, 8b, 9b, 10a and 11.

Conclusion

Puripotent human H7 and mouse F9 embryonal cells express the genes for most of the Wnts and Frizzleds. In contrast, multipotent human mesenchymal cells are deficient in expression of Frizzled-8 and of 6 Wnt genes.     

Anterior cruciate ligament constructs fabricated from human mesenchymal stem cells in a collagen type I hydrogel

BACKGROUND: Disruptions of the anterior cruciate ligament (ACL) of the knee joint are common and are currently treated using ligament or tendon grafts. In this study, we tested the hypothesis that it is possible to fabricate an ACL construct in vitro using mesenchymal stem cells (MSC) in combination with an optimized collagen type I hydrogel, which is in clinical use for autologous chondrocyte transplantation (ACT). METHODS: ACL constructs were molded using a collagen type I hydrogel containing 5 x 10(5) MSC/mL and non-demineralized bone cylinders at each end of the constructs. The constructs were kept in a horizontal position for 10 days to allow the cells and the gel to remodel and attach to the bone cylinders. Thereafter, cyclic stretching with 1 Hz was performed for 14 days (continuously for 8 h/day) in a specially designed bioreactor. RESULTS: Histochemical analysis for H and E, Masson-Goldner and Azan and immunohistochemical analysis for collagen types I and III, fibronectin and elastin showed elongated fibroblast-like cells embedded in a wavy orientated collagenous tissue, together with a ligament-like extracellular matrix in the cyclic stretched constructs. No orientation of collagen fibers and cells, and no formation of a ligament-like matrix, could be seen in the non-stretched control group cultured in a horizontal position without tension. RT-PCR analysis revealed an increased gene expression of collagen types I and III, fibronectin and elastin in the stretched constructs compared with the non-stretched controls. DISCUSSION: In conclusion, ACL-like constructs from a collagen type I hydrogel, optimized for the reconstruction of ligaments, and MSC have been fabricated. As shown by other investigators, who analyzed the influence of cyclic stretching on the differentiation of MSC, our results indicate a ligament-specific increased protein and gene expression and the formation of a ligament-like extracellular matrix. The fabricated constructs are still too weak for animal experiments or clinical application and current investigations are focusing on the development of a construct with an internal augmentation using biodegradable fibers.     

Primary mesenchymal stem and progenitor cells from bone marrow lack expression of CD44 protein

Despite significant progress in our understanding of mesenchymal stem cell (MSC) biology during recent years, much of the information is based on experiments using in vitro culture-selected stromal progenitor cells. Therefore, the natural cellular identity of MSCs remains poorly defined. Numerous studies have reported that CD44 expression is one of the characteristics of MSCs in both humans and mice; however, we here have prospectively isolated bone marrow stromal cell subsets from both human and mouse bone marrow by flow cytometry and characterized them by gene expression analysis and function assays. Our data provide functional and molecular evidence suggesting that primary mesenchymal stem and progenitor cells of bone marrow reside in the CD44(-) cell fraction in both mice and humans. The finding that these CD44(-) cells acquire CD44 expression after in vitro culture provides an explanation for the previous misconceptions concerning CD44 expression on MSCs. In addition, the other previous reported MSC markers, including CD73, CD146, CD271, and CD106/VCAM1, are also differentially expressed on those two cell types. Our microarray data revealed a distinct gene expression profile of the freshly isolated CD44(-) cells and the cultured MSCs generated from these cells. Thus, we conclude that bone marrow MSCs physiologically lack expression of CD44, highlighting the natural phenotype of MSCs and opening new possibilities to prospectively isolate MSCs from the bone marrow.     

Differential expression of microRNAs in decidua-derived mesenchymal stem cells from patients with pre-eclampsia

Background

Mesenchymal stem cells (MSCs) at maternal-fetal interface are considered to play an important role in the pathogenesis of pre-eclampsia (PE). microRNAs (miRNAs) also have an important influence on differentiation, maturation, and functions of MSCs. Our aim in this study was to determine the differential expression of miRNAs in decidua-derived MSCs (dMSCs) from severe PE and normal pregnancies.

Results

miRNA expression profiles in dMSCs from five patients with severe PE and five healthy pregnant women were screened using microarray. Then, bioinformatic analysis of the microarray results was performed. Out of 179 differentially expressed miRNAs, 49 miRNAs had significant (p < 0.05) differential expression of ≥ 2.0-fold changes, including 21 up-regulated and 28 down-regulated. miRNA-Gene-network and miRNA-Gene ontology (GO) -network analyses were performed. Overall, 21 up-regulated and 15 down-regulated miRNAs showed high degrees in these analyses. Moreover, the significantly enriched signaling pathways and GOs were identified. The analyses revealed that pathways associated with cell proliferation, angiogenesis, and immune functions were highly regulated by the differentially expressed miRNAs, including Wnt signaling pathway, mitogen-activated protein kinase signaling pathway, transforming growth factor beta signaling pathway, T-cell receptor signaling pathway, and B cell receptor signaling pathway. Four miRNA predicted target genes, vascular endothelial growth factor A (VEGFA), indoleamine 2,3-dioxygenase, suppression of cytokine signaling 3, and serine/threonine protein phosphatase 2A 55 kDa regulatory subunit B α isoform (PPP2R2A) were all decreased in dMSCs from patients with PE. Furthermore, the physiological roles of miR-16 and miR-136 in the down-regulation of VEGFA and PPP2R2A, respectively, were confirmed through reporter assays.

Conclusions

These findings suggest that miRNAs in dMSCs may be important regulatory molecules in the development of PE.     

Restoration of SMN expression in mesenchymal stem cells derived from gene-targeted patient-specific iPSCs

Spinal muscular atrophy (SMA) is primarily a neurodegenerative disease caused by the homozygous deletion of the survival motor neuron 1 (SMN1) gene, thereby reducing SMN protein expression. Mesenchymal stem cells (MSCs) have been implicated in the treatment of SMA. In the present study, we overexpressed exogenous SMN1 at the ribosomal DNA (rDNA) locus of induced pluripotent stem cells (iPSCs) generated from a SMA patient using an rDNA-targeting vector. The gene-targeted patient iPSCs differentiated into MSCs (SMN1-MSCs). A 2.1-fold higher expression level of SMN protein was detected in SMN1-MSCs than that detected in MSCs derived from patient iPSCs, and the results of the immunofluorescence analysis showed no difference in the quantity of SMN nuclear structures (gems) between SMN1-MSCs and MSCs derived from normal human iPSCs (h-MSCs). These findings provide a novel strategy for obtaining gene-targeted MSCs for potential clinical applications in autologous cell-based therapy.     

Dexamethasone downregulates the expression of parathyroid hormone-related protein (PTHrP) in mesenchymal stem cells

Parathyroid hormone-related protein (PTHrP) has been shown to have anabolic effects in women with postmenopausal osteoporosis. PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The receptor responsible for the effects of PTHrP is the common PTH/PTHrP receptor (PTH1R). Glucocorticoids (GC) are commonly used as drugs to treat inflammatory diseases. Long-term GC treatments are often associated with bone loss which can lead to GC-induced osteoporosis. The aim of this work was to study the effects of the glucocorticoid dexamethasone (Dex) on the expression of PTHrP and PTH1R in adult human mesenchymal stem cells, the progenitor cells of osteoblasts.Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The expression of PTHrP and PTH1R mRNA was assayed by real-time qPCR. The PTHrP release into the culture media was measured by an immunoradiometric assay.Treatment with Dex (10 nM) resulted in an 80% drop in the PTHrP release within 6 h. A 24 h Dex treatment also reduced the expression of PTHrP mRNA by up to 90%. The expression of PTH1R receptor mRNA was simultaneously increased up to 20-fold by 10 nM Dex. The effects of Dex on PTHrP and PTH1R were dose-dependent and experiments with the GC-receptor antagonist mifepristone showed an involvement of GC-receptors in these effects. In addition to the Dex-induced effects on PTHrP and PTH1R, Dex also increased mineralization and the expression of the osteoblast markers Runx2 and alkaline phosphatase. In our studies, we show that dexamethasone decreases the expression of PTHrP and increases the expression of the PTH1R receptor. This could have an impact on PTHrP-mediated anabolic actions on bone and could also affect the responsiveness of circulating PTH. The results indicate that glucocorticoids affect the signalling pathway of PTHrP by regulating both PTHrP and PTH1R expression and these mechanisms could be involved in glucocorticoid-induced osteoporosis.     

Wnt inhibitors enhance chondrogenesis of human mesenchymal stem cells in a long-term pellet culture

The long-term effects (~3 weeks) of two Wnt inhibitors (dickkopf [DKK]-1 and secreted frizzled-related protein [sFRP]-1), on the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) was determined. Wnt inhibitors significantly increased the amount of glycosaminoglycan (GAG) in treated pellets (P < 0.05). The gene expression of COL2A1 increased and COL1A1 decreased while the gene expression of SOX-9 and COL10A1 did not change significantly after three weeks of in vitro culture. The protein expression of type II collagen significantly increased (P < 0.05) and that of type I collagen significantly decreased (P < 0.05) while SOX-9 and type X collagen protein expression was unaffected. These findings suggest that Wnt inhibitors promote the chondrogenic differentiation of hMSCs when treated for three weeks.     

Wnt3a promotes differentiation of human bone marrow-derived mesenchymal stem cells into cementoblast-like cells

Cementum is a calcified, avascular connective tissue that laminates the root of a tooth and plays a pivotal role in the development, homeostasis, and regeneration of a periodontal tissue. As a potential treatment for periodontal tissue defects in the patient with chronic periodontitis, much attention has been paid to tissue engineering combined with mesenchymal stem cells for regenerating periodontal tissues including cementum. However, limited information is available for the molecular factors that have impacts on the differentiation of mesenchymal stem cells into cementoblasts. Here, we focus on the effect of Wnt3a as a potential inducer and tested the effect of this protein in vitro using human bone marrow-derived mesenchymal stem cells. It was found that, when cells were cultured in an osteogenic medium containing Wnt3a, cementoblast-specific genes, such as cementum protein 1 and cementum attachment protein, as well as bone-related genes were significantly upregulated. These results suggest that Wnt3a promotes differentiation of the cells into cementoblast-like cells. Further experiments were carried out using inhibitors to gain deeper insights into molecular mechanisms underlying the observed differentiation. As a result, we conclude that Wnt3a-triggered differentiation into cementoblast-like cells is the consequence of the activation of the canonical Wnt signaling pathway with possible involvement of the non-canonical pathway.     

Wnt signaling in stem and cancer stem cells

Canonical Wnt signaling supports the formation and maintenance of stem and cancer stem cells. Recent studies have elucidated epigenetic mechanisms that control pluripotency and stemness, and allow a first assessment how embryonic and tissue stem cells are generated and maintained, and how Wnt signaling might be involved. The core of this review highlights the roles of Wnt signaling in stem and cancer stem cells of tissues such as skin, intestine and mammary gland. Lastly, we refer to the characterization of novel and powerful inhibitors of canonical Wnt signaling and describe attempts to bring these compounds into preclinical and clinical studies.     

Aspirin induces apoptosis in mesenchymal stem cells requiring Wnt/β-catenin pathway

Background and Objectives:  Mesenchymal stem cells (MSC) are multipotent progenitor cells that are have found use in regenerative medicine. We have previously observed that aspirin, a widely used anti-inflammatory drug, inhibits MSC proliferation. Here we have aimed to elucidate whether aspirin induces MSC apoptosis and whether this is modulated through the Wnt/β-catenin pathway.
Materials and methods:  Apoptosis of MSCs was assessed using Hoechst 33342 dye and an Annexin V–FITC/PI Apoptosis Kit. Expression of protein and protein phosphorylation were investigated using Western blot analysis. Caspase-3 activity was detected by applying a caspase-3/CPP32 Colorimetric Assay Kit.
Results:  In these MSCs, aspirin induced morphological changes characteristic of apoptosis, cytochrome  c release from mitochondria, and caspase-3 activation. Stimulating the Wnt/β-catenin pathway by both Wnt 3a and GSK-3β inhibitors (LiCl and SB 216763), blocked aspirin-induced apoptosis and protected mitochondrial function, as demonstrated by decreased cytochrome  c release and caspase-3 activity. Aspirin initially caused a time-dependent decrease in COX-2 expression but subsequently, and unexpectedly, elevated the latter. Stimulation of COX-2 expression by aspirin was further enhanced following stimulation of the Wnt/β-catenin pathway. Application of the COX-2 inhibitor NS-398 suppressed elevated COX-2 expression and promoted aspirin-induced apoptosis.
Conclusion:  These results demonstrate that the Wnt/β-catenin pathway is a key modulator of aspirin-induced apoptosis in MSCs by regulation of mitochrondrial/caspase-3 function. More importantly, our findings suggest that aspirin may influence MSC survival under certain conditions; therefore, it should be used with caution when considering regenerative MSC transplantation in patients with concomitant chronic inflammatory diseases such as arthritis.     

Exosomes from human adipose-derived mesenchymal stem cells promote migration through Wnt signaling pathway in a breast cancer cell model

Recent studies have demonstrated that the Wnt/β-catenin signaling plays an important role in stem cell aging. However, the mechanisms of cell senescence induced by Wnt/β-catenin signaling are still poorly understood. Our preliminary study has indicated that activated Wnt/β-catenin signaling can induce MSC aging. In this study, we reported that the Wnt/β-catenin signaling was a potent activator of reactive oxygen species (ROS) generation in MSCs. After scavenging ROS with N-acetylcysteine, Wnt/β-catenin signaling-induced MSC aging was significantly attenuated and the DNA damage and the expression of p16^INK4A, p53, and p21 were reduced in MSCs. These results indicated that the Wnt/β-catenin signaling could induce MSC aging through promoting the intracellular production of ROS, and ROS may be the main mediators of MSC aging induced by excessive activation of Wnt/β-catenin signaling.     

Zinc finger protein 521 suppresses osteogenic differentiation of rat mesenchymal stem cells by inhibiting the Wnt/beta-catenin signaling pathway

Zinc finger protein 521 (Zfp521) is involved in a number of cellular processes in a variety of cells and tissues. In the present study, the effects of Zfp521 on osteogenic differentiation of rat mesenchymal stem cells (MSCs) were investigated. The results showed that, in rat MSCs, knocking down cellular Zfp521 by short hairpin RNA (shRNA) decreases cell proliferation while promoting ALP activity, calcium accumulation, and the expression of mRNA that encodes bone sialoprotein (BSP), osteocalcin (OCN) and Runx2. Furthermore, in Zfp521-depleted cells, the up-regulation of phospho-Wnt (p-Wnt) and beta-catenin expression levels was detected. However, over-expression of Zfp521 played the opposite role in proliferation and osteogenic differentiation of rat MSCs. To further demonstrate the functions of the Wnt/beta-catenin signaling in Zfp521 regulated-osteogenic differentiation, the activation of Wnt/beta-catenin was blocked with IWP-2 inhibitor. The suppression of the Wnt/beta-catenin pathway completely abrogated the effects of Zfp521 knockdown on osteogenic differentiation of rat MSCs. Therefore, we conclude that Zfp521 regulates osteogenic differentiation of rat MSCs through the suppression of the Wnt/beta-catenin signaling pathway.     

Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells

The presence within bone marrow of a population of mesenchymal stem cells (MSCs) able to differentiate into a number of different mesenchymal tissues, including bone and cartilage, was first suggested by Friedenstein nearly 40 years ago. Since then MSCs have been demonstrated in a variety of fetal and adult tissues, including bone marrow, fetal blood and liver, cord blood, amniotic fluid and, in some circumstances, in adult peripheral blood. MSCs from all of these sources can be extensively expanded in vitro and when cultured under specific permissive conditions retain their ability to differentiate into multiple lineages including bone, cartilage, fat, muscle, nerve, glial and stromal cells. There has been great interest in these cells both because of their value as a model for studying the molecular basis of differentiation and because of their therapeutic potential for tissue repair and immune modulation. However, MSCs are a rare population in these tissues. Here we tried to identify cells with MSC-like potency in human placenta. We isolated adherent cells from trypsin-digested term placentas and examined these cells for morphology, surface markers, and differentiation potential and found that they expressed several stem cell markers. They also showed endothelial and neurogenic differentiation potentials under appropriate conditions. We suggest that placenta-derived cells have multilineage differentiation potential similar to MSCs in terms of morphology and cell-surface antigen expression. The placenta may prove to be a useful source of MSCs.     

Cytokine interactions in mesenchymal stem cells from cord blood

We used cytokine protein array to analyze the expression of cytokines from human cord blood-derived mesenchymal stem cells (CB-MSCs). Several cytokines, interleukins (IL), and growth factors, including ENA-78, GM-CSF, GRO, IL-1β, IL-6, IL-8, MCP-1, OSM, VEGF, FGF-4, FGF-7, FGF-9, GCP-2, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IP-10, LIF, MIF, MIP-3α, osteoprotegerin, PARC, PIGF, TGF-β2, TGF-β3, TIMP-1, as well as TIMP-2, were secreted by CB-MSCs, while IL-4, IL-5, IL-7, IL-13, TGF-β1, TNF-α, and TNF-β were not expressed under normal growth conditions. IL-6, IL-8, TIMP-1, and TIMP-2 were the most abundant interleukins expressed by CB-MSCs. A set of growth factors were selected to evaluate their stimulatory effects on the IL6 secretion for CB-MSCs. IL-1β was the most important factor inducing CB-MSC to secret IL-6. The mechanism by which IL-1β promoted IL-6 expression in CB-MSCs was studied. By using various inhibitors of signal transduction, we found that activation of p38 mitogen-activated protein kinases (MAPK) and MAPK kinase (MEK) is essential in the IL-1β stimulated signaling cascade which leads to the increase in IL-6 synthesis. Additionally, continuous supplement of IL-1β in the CB-MSCs culture will facilitate adipogenic maturation of CB-MSCs as evidenced by the presence of oil drops in the CB-MSCs and secretion of leptin, a molecule marker of adipocytes. These results strongly suggest that cytokine induction and signal transduction are important for the differentiation of CB-MSCs.