RhoA/ROCK, cytoskeletal dynamics, and focal adhesion kinase are required for mechanical stretch-induced tenogenic differentiation of human mesenchymal stem cells

Human bone marrow mesenchymal stem cells (hMSCs) have the potential to differentiate into tendon/ligament-like lineages when they are subjected to mechanical stretching. However, the means through which mechanical stretch regulates the tenogenic differentiation of hMSCs remains unclear. This study examined the role of RhoA/ROCK, cytoskeletal organization, and focal adhesion kinase (FAK) in mechanical stretch-induced tenogenic differentiation characterized by the up-regulation of tendon-related marker gene expression. Our findings showed that RhoA/ROCK and FAK regulated mechanical stretch-induced realignment of hMSCs by regulating cytoskeletal organization and that RhoA/ROCK and cytoskeletal organization were essential to mechanical stretch-activated FAK phosphorylation at Tyr397. We also demonstrated that this process can be blocked by Y-27632 (a specific inhibitor of RhoA/ROCK), cytochalasin D (an inhibitor of cytoskeletal organization) or PF 573228 (a specific inhibitor of FAK). The results of this study suggest that RhoA/ROCK, cytoskeletal organization, and FAK compose a "signaling network" that senses mechanical stretching and drives mechanical stretch-induced tenogenic differentiation of hMSCs. This work provides novel insights regarding the mechanisms of tenogenesis in a stretch-induced environment and supports the therapeutic potential of hMSCs.     

Aspirin promotes tenogenic differentiation of tendon stem cells and facilitates tendinopathy healing through regulating the GDF7/Smad1/5 signaling pathway

Tendinopathy is a common musculoskeletal system disorder in sports medicine, but regeneration ability of injury tendon is limited. Tendon stem cells (TSCs) have shown the definitive treatment evidence for tendinopathy and tendon injuries due to their tenogenesis capacity. Aspirin, as the representative of nonsteroidal anti-inflammatory drugs for its anti-inflammatory and analgestic actions, has been commonly used in treating tendinopathy in clinical, but the effect of aspirin on tenogenesis of TSCs is unclear. We hypothesized that aspirin could promote injury tendon healing through inducing TSCs tenogenesis. The aim of the present study is to make clear the effect of aspirin on TSC tenogenesis and tendon healing in tendinopathy, and thus provide new treatment evidence and strategy of aspirin for clinical practice. First, TSCs were treated with aspirin under tenogenic medium for 3, 7, and 14 days. Sirius Red staining was performed to observe the TSC differentiation. Furthermore, RNA sequencing was utilized to screen out different genes between the induction group and aspirin treatment group. Then, we identified the filtrated molecules and compared their effect on tenogenesis and related signaling pathway. At last, we constructed the tendinopathy model and compared biomechanical changes after aspirin intake. From the results, we found that aspirin promoted tenogenesis of TSCs. RNA sequencing showed that growth differentiation factor 6 (GDF6), GDF7, and GDF11 were upregulated in induction medium with the aspirin group compared with the induction medium group. GDF7 increased tenogenesis and activated Smad1/5 signaling. In addition, aspirin increased the expression of TNC, TNMD, and Scx and biomechanical properties of the injured tendon. In conclusion, aspirin promoted TSC tenogenesis and tendinopathy healing through GDF7/Smad1/5 signaling, and this provided new treatment evidence of aspirin for tendinopathy and tendon injuries.     

Radiation-Induced Alterations of Osteogenic and Chondrogenic Differentiation of Human Mesenchymal Stem Cells

While human mesenchymal stem cells (hMSCs), either in the bone marrow or in tumour microenvironment could be targeted by radiotherapy, their response is poorly understood. The oxic effects on radiosensitivity, cell cycle progression are largely unknown, and the radiation effects on hMSCs differentiation capacities remained unexplored. Here we analysed hMSCs viability and cell cycle progression in 21% O₂ and 3% O₂ conditions after medical X-rays irradiation. Differentiation towards osteogenesis and chondrogenesis after irradiation was evaluated through an analysis of differentiation specific genes. Finally, a 3D culture model in hypoxia was used to evaluate chondrogenesis in conditions mimicking the natural hMSCs microenvironment. The hMSCs radiosensitivity was not affected by O₂ tension. A decreased number of cells in S phase and an increase in G2/M were observed in both O₂ tensions after 16 hours but hMSCs released from the G2/M arrest and proliferated at day 7. Osteogenesis was increased after irradiation with an enhancement of mRNA expression of specific osteogenic genes (alkaline phosphatase, osteopontin). Osteoblastic differentiation was altered since matrix deposition was impaired with a decreased expression of collagen I, probably through an increase of its degradation by MMP-3. After induction in monolayers, chondrogenesis was altered after irradiation with an increase in COL1A1 and a decrease in both SOX9 and ACAN mRNA expression. After induction in a 3D culture in hypoxia, chondrogenesis was altered after irradiation with a decrease in COL2A1, ACAN and SOX9 mRNA amounts associated with a RUNX2 increase. Together with collagens I and II proteins decrease, associated to a MMP-13 expression increase, these data show a radiation-induced impairment of chondrogenesis. Finally, a radiation-induced impairment of both osteogenesis and chondrogenesis was characterised by a matrix composition alteration, through inhibition of synthesis and/or increased degradation. Alteration of osteogenesis and chondrogenesis in hMSCs could potentially explain bone/joints defects observed after radiotherapy.     

Circadian Clock Genes Modulate Human Bone Marrow Mesenchymal Stem Cell Differentiation,Migration and Cell Cycle

Many of the components that regulate the circadian clock have been identified in organisms and humans. The influence of circadian rhythm (CR) on the regulation of stem cells biology began to be evaluated. However, little is known on the role of CR on human mesenchymal stem cell (hMSCs) properties. The objective of this study was to investigate the influence of CR on the differentiation capacities of bone marrow hMSCs, as well as the regulation of cell cycle and migration capabilities. To that, we used both a chemical approach with a GSK-3β specific inhibitor (2’E,3’Z-6-bromoindirubin-3’-oxime, BIO) and a knockdown of CLOCK and PER2, two of the main genes involved in CR regulation. In these experimental conditions, a dramatic inhibition of adipocyte differentiation was observed, while osteoblastic differentiation capacities were not modified. In addition, cell migration was decreased in PER2^-/- cells. Lastly, downregulation of circadian clock genes induced a modification of the hMSCs cell cycle phase distribution, which was shown to be related to a change of the cyclin expression profile. Taken together, these data showed that CR plays a role in the regulation of hMSCs differentiation and division, and likely represent key factor in maintaining hMSCs properties.     

Endothelial Differentiation of Mesenchymal Stromal Cells

Human mesenchymal stromal cells (hMSCs) are increasingly used in regenerative medicine for restoring worn-out or damaged tissue. Newly engineered tissues need to be properly vascularized and current candidates for in vitro tissue pre-vascularization are endothelial cells and endothelial progenitor cells. However, their use in therapy is hampered by their limited expansion capacity and lack of autologous sources. Our approach to engineering large grafts is to use hMSCs both as a source of cells for regeneration of targeted tissue and at the same time as the source of endothelial cells. Here we investigate how different stimuli influence endothelial differentiation of hMSCs. Although growth supplements together with shear force were not sufficient to differentiate hMSCs with respect to expression of endothelial markers such as CD31 and KDR, these conditions did prime the cells to differentiate into cells with an endothelial gene expression profile and morphology when seeded on Matrigel. In addition, we show that endothelial-like hMSCs are able to create a capillary network in 3D culture both in vitro and in vivo conditions. The expansion phase in the presence of growth supplements was crucial for the stability of the capillaries formed in vitro. To conclude, we established a robust protocol for endothelial differentiation of hMSCs, including an immortalized MSC line (iMSCs) which allows for reproducible in vitro analysis in further studies.     

生长分化因子15及其特异性受体GFRAL信号通路

生长分化因子 15(growth differentiation factor 15,GDF15)属于转化生长因子 β(transforming growth factor β,TGF-β)超家族的成员之一,是与转化生长因子β家族成员同源性很低的新一类二聚体多肽.GDF15最初发现于活化的巨噬细胞中,可通过2种不同的细...     

GDF1 is a novel mediator of macrophage infiltration in brown adipose tissue of obese mice

We previously demonstrated a marked upregulation in the bone morphogenic protein (BMP)/growth differentiation factor (GDF) family member, GDF5, which is capable of promoting brown adipogenesis, in brown adipose tissue (BAT) of obese mice. In this study, we identified other GDF family members, besides GDF5 that are responsive to different obesogenic signals in BAT using inborn and acquired obesity animal models. In BAT from leptin-deficient ob/ob mice, GDF1 expression was preferentially downregulated, whereas the expression of several other genes in the BMP/GDF family, including GDF5, was upregulated. Moreover, in cultured brown adipocytes exposed to tunicamycin and hydrogen peroxide, at concentrations not affecting cellular viability, GDF1 expression was significantly downregulated. Recombinant GDF1 failed to significantly alter brown adipogenesis, despite the promoted phosphorylation of Smad1/5/8 in cultured brown adipocytes, but accelerated Smad1/5/8 phosphorylation with a concomitant increase in the number of migrating cells during exposure in a manner sensitive to activin-like kinase inhibitors in macrophagic RAW264.7 cells. Similarly, accelerated migration was observed in murine peritoneal macrophages exposed to GDF1. These results indicate that obesity could lead to predominant downregulation of GDF1 expression in BAT, which can modulate cellular migration through a mechanism relevant to activation of the downstream Smad signaling pathway in adjacent macrophages.     

Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells

Several biological events are controlled by Hedgehog (Hh) signaling, including osteoblast phenotype development. This study aimed at evaluating the gene expression profile of human mesenchymal stem cells (hMSCs) treated with the Hh agonist, purmorphamine, focusing on Hh signaling and osteoblast differentiation. hMSCs from bone marrow were cultured in non‐osteogenic medium with or without purmorphamine (2 µM) for periods of up to 14 days. Purmorphamine up‐regulated gene expression of the mediators of Hh pathway, SMO, PTCH1, GLI1, and GLI2. The activation of Hh pathway by purmorphamine increased the expression of several genes (e.g., RUNX2 and BMPs) related to osteogenesis. Our results indicated that purmorphamine triggers Hh signaling pathway in hMSCs, inducing an increase in the expression of a set of genes involved in the osteoblast differentiation program. Thus, we conclude that Hh is a crucial pathway in the commitment of undifferentiated cells to the osteoblast lineage. J. Cell. Biochem. 113: 204–208, 2012. © 2011 Wiley Periodicals, Inc.     

Gene Expression Profiles of Human Mesenchymal Stromal Cells Derived from Wharton’s Jelly and Amniotic Membrane before and after Osteo-Induction Using NanoString Platform

The use of perinatal mesenchymal stem cells (MSCs) in bone tissue regeneration and engineering to substitute bone marrow MSCs has drawn great interest due to their high yield, ease of procurement, multilineage differentiation potential and lack of ethical concerns. Although amniotic membrane (AM) and Wharton’s jelly (WJ)-derived MSCs have been widely shown to possess osteogenic differentiation potential, the intrinsic properties determining their osteogenic capacity remain unclear. Here, we compared gene expression profiles of AM- and WJ-MSCs at basal and osteogenic conditions by using the NanoString Stem Cell Panel containing regulatory genes associated with stemness, self-renewal, Wnt, Notch and Hedgehog signalling pathways. At basal condition, WJ-MSCs displayed higher expression in most genes regardless of their functional roles in self-renewal, adhesion, or differentiation signalling pathways. After osteo-induction, elevated expression of self-renewal genes ADAR and PAFAH1B1 was observed in AM-MSCs, while stemness genes MME and ALDH1A1 were upregulated in WJ-MSC. Both MSCs showed differences in genes associated with ligands, receptors and ubiquitin ligases of the Notch pathway. In addition, further evidence was demonstrated in some signalling molecules including CTBPs, protein kinases, phosphatases, RHOA, RAC1. Downstream targets HES1 and JUN especially showed higher expression in non-induced WJ-MSCs. Hedgehog genes initially expressed in both MSCs were downregulated in WJ-MSCs during osteogenesis. This study has provided insights into the intrinsic biological differences that may lead to their discrimination in therapeutic intervention.     

Wnts Enhance Neurotrophin-Induced Neuronal Differentiation in Adult Bone-Marrow-Derived Mesenchymal Stem Cells via Canonical and Noncanonical Signaling Pathways

Wnts were previously shown to regulate the neurogenesis of neural stem or progenitor cells. Here, we explored the underlying molecular mechanisms through which Wnt signaling regulates neurotrophins (NTs) in the NT-induced neuronal differentiation of human mesenchymal stem cells (hMSCs). NTs can increase the expression of Wnt1 and Wnt7a in hMSCs. However, only Wnt7a enables the expression of synapsin-1, a synaptic marker in mature neurons, to be induced and triggers the formation of cholinergic and dopaminergic neurons. Human recombinant (hr)Wnt7a and general neuron makers were positively correlated in a dose- and time-dependent manner. In addition, the expression of synaptic markers and neurites was induced by Wnt7a and lithium, a glycogen synthase kinase-3β inhibitor, in the NT-induced hMSCs via the canonical/β-catenin pathway, but was inhibited by Wnt inhibitors and frizzled-5 (Frz5) blocking antibodies. In addition, hrWnt7a triggered the formation of cholinergic and dopaminergic neurons via the non-canonical/c-jun N-terminal kinase (JNK) pathway, and the formation of these neurons was inhibited by a JNK inhibitor and Frz9 blocking antibodies. In conclusion, hrWnt7a enhances the synthesis of synapse and facilitates neuronal differentiation in hMSCS through various Frz receptors. These mechanisms may be employed widely in the transdifferentiation of other adult stem cells.     

Integrated analyses of copy number variations and gene expression in lung adenocarcinoma

Numerous efforts have been made to elucidate the etiology and improve the treatment of lung cancer, but the overall five-year survival rate is still only 15%. Identification of prognostic biomarkers for lung cancer using gene expression microarrays poses a major challenge in that very few overlapping genes have been reported among different studies. To address this issue, we have performed concurrent genome-wide analyses of copy number variation and gene expression to identify genes reproducibly associated with tumorigenesis and survival in non-smoking female lung adenocarcinoma. The genomic landscape of frequent copy number variable regions (CNVRs) in at least 30% of samples was revealed, and their aberration patterns were highly similar to several studies reported previously. Further statistical analysis for genes located in the CNVRs identified 475 genes differentially expressed between tumor and normal tissues (p<10⁻⁵). We demonstrated the reproducibility of these genes in another lung cancer study (p = 0.0034, Fisher''s exact test), and showed the concordance between copy number variations and gene expression changes by elevated Pearson correlation coefficients. Pathway analysis revealed two major dysregulated functions in lung tumorigenesis: survival regulation via AKT signaling and cytoskeleton reorganization. Further validation of these enriched pathways using three independent cohorts demonstrated effective prediction of survival. In conclusion, by integrating gene expression profiles and copy number variations, we identified genes/pathways that may serve as prognostic biomarkers for lung tumorigenesis.     

Adenoviral Vectors Stimulate Glucagon Transcription in Human Mesenchymal Stem Cells Expressing Pancreatic Transcription Factors

Viral gene carriers are being widely used as gene transfer systems in (trans)differentiation and reprogramming strategies. Forced expression of key regulators of pancreatic differentiation in stem cells, liver cells, pancreatic duct cells, or cells from the exocrine pancreas, can lead to the initiation of endocrine pancreatic differentiation. While several viral vector systems have been employed in such studies, the results reported with adenovirus vectors have been the most promising in vitro and in vivo. In this study, we examined whether the viral vector system itself could impact the differentiation capacity of human bone-marrow derived mesenchymal stem cells (hMSCs) toward the endocrine lineage. Lentivirus-mediated expression of Pdx-1, Ngn-3, and Maf-A alone or in combination does not lead to robust expression of any of the endocrine hormones (i.e. insulin, glucagon and somatostatin) in hMSCs. Remarkably, subsequent transduction of these genetically modified cells with an irrelevant early region 1 (E1)-deleted adenoviral vector potentiates the differentiation stimulus and promotes glucagon gene expression in hMSCs by affecting the chromatin structure. This adenovirus stimulation was observed upon infection with an E1-deleted adenovirus vector, but not after exposure to helper-dependent adenovirus vectors, pointing at the involvement of genes retained in the E1-deleted adenovirus vector in this phenomenon. Lentivirus mediated expression of the adenovirus E4-ORF3 mimics the adenovirus effect. From these data we conclude that E1-deleted adenoviral vectors are not inert gene-transfer vectors and contribute to the modulation of the cellular differentiation pathways.     

TGF-β regulates β-catenin signaling and osteoblast differentiation in human mesenchymal stem cells

Human adult bone marrow-derived skeletal stem cells a.k.a mesenchymal stem cells (hMSCs) have been shown to be precursors of several different cellular lineages, including osteoblast, chondrocyte, myoblast, adipocyte, and fibroblast. Several studies have shown that cooperation between transforming growth factor β (TGF-β) and Wnt/β-catenin signaling pathways plays a role in controlling certain developmental events and diseases. Our previous data showed that agents like TGF-β, cooperation with Wnt signaling, promote chondrocyte differentiation at the expense of adipocyte differentiation in hMSCs. In this study, we tested mechanisms by which TGF-β activation of β-catenin signaling pathway and whether these pathways interact during osteoblast differentiation of hMSCs. With selective small chemical kinase inhibitors, we demonstrated that TGF-β1 requires TGF-β type I receptor ALK-5, Smad3, phosphoinositide 3-kinases (PI3K), and protein kinase A (PKA) to stabilize β-catenin, and needs ALK-5, PKA, and JNK to inhibit osteoblastogenesis in hMSCs. Knockdown of β-catenin with siRNA stimulated alkaline phosphatase activity and antagonized the inhibitory effects of TGF-β1 on bone sialoprotein (BSP) expression, suggested that TGF-β1 cooperated with β-catenin signaling in inhibitory of osteoblastogenesis in hMSCs. In summary, TGF-β1 activates β-catenin signaling pathway via ALK-5, Smad3, PKA, and PI3K pathways, and modulates osteoblastogenesis via ALK5, PKA, and JNK pathways in hMSCs; the interaction between TGF-β and β-catenin signaling supports the view that β-catenin signaling is a mediator of TGF-β's effects on osteoblast differentiation of hMSCs.     

Tenogenic differentiation of equine adipose-tissue-derived stem cells under the influence of tensile strain, growth differentiation factors and various oxygen tensions

Mesenchymal stem cells have become extremely interesting for regenerative medicine and tissue engineering in the horse. Stem cell therapy has been proven to be a powerful and successful instrument, in particular for the healing of tendon lesions. We pre-differentiated equine adipose-tissue-derived stem cells (ASCs) in a collagen I gel scaffold by applying tensile strain, growth differentiation factors (GDFs) and various oxygen tensions in order to determine the optimal conditions for in vitro differentiation toward the tenogenic lineage. We compared the influence of 3% versus 21% oxygen tension, the use of GDF 5, GDF 6 and GDF 7 and the application of uniaxial tensile strain versus no mechanical stimulation on differentiation results as evaluated by cell morphology and by the expression of the tendon-relevant genes collagen I, collagen III, cartilage oligomeric matrix protein and scleraxis. The best results were obtained with an oxygen tension of 21%, tensile stimulation and supplementation with GDF 5 or GDF 7. This approach raises the hope that the in vivo application of pre-differentiated stem cells will improve healing and recovery time in comparison with treatment involving undifferentiated stem cells.     

FGF-2 increases osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells by inactivation of TGF-β signaling

Human mesenchymal stem cells (hMSCs) are able to self-replicate and differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, endothelial cells, and muscle cells. It was reported that fibroblast growth factor-2 (FGF-2) increased the growth rate and multidifferentiation potentials of hMSCs. In this study, we investigated the genes involved in the promotion of osteogenic and chondrogenic differentiation potentials of hMSCs in the presence of FGF-2. hMSCs were maintained in the medium with FGF-2. hMSCs were harvested for the study of osteogenic or chondrogenic differentiation potential after 15 days’ culture. To investigate osteogenic differentiation, the protein levels of alkaline phosphatase (ALP) and the mRNA expression levels of osteocalcin were measured after the induction of osteogenic differentiation. Moreover, the investigation for chondrogenic differentiation was performed by measuring the mRNA expression levels of type II and type X collagens after the induction of chondrogenic differentiation. The expression levels of ALP, type II collagen, and type X collagen of hMSCs cultured with FGF-2 were significantly higher than control. These results suggested that FGF-2 increased osteogenic and chondrogenic differentiation potentials of hMSCs. Furthermore, microarray analysis was performed after 15 days’ culture in the medium with FGF-2. We found that the overall insulin-like growth factor-I (IGF-I) and transforming growth factor-β (TGF-β) signaling pathways were inactivated by FGF-2. These results suggested that the inactivation of IGF-I and TGF-β signaling promotes osteogenic and chondrogenic differentiation potential of hMSCs in the presence of FGF-2.