Fate Of Mesenchymal Stem Cells Research Articles

A computational model to explore the role of angiogenic impairment on endochondral ossification during fracture healing.

While it is well established that an adequate blood supply is critical to successful bone regeneration, it remains poorly understood how progenitor cell fate is affected by the altered conditions present in fractures with disrupted vasculature. In this study, computational models were used to explore how angiogenic impairment impacts oxygen availability within a fracture callus and hence regulates mesenchymal stem cell (MSC) differentiation and bone regeneration. Tissue differentiation was predicted using a previously developed algorithm which assumed that MSC fate is governed by oxygen tension and substrate stiffness. This model was updated based on the hypothesis that cell death, chondrocyte hypertrophy and endochondral ossification are regulated by oxygen availability. To test this, the updated model was used to simulate the time course of normal fracture healing, where it successfully predicted the observed quantity and spatial distribution of bone and cartilage at 10 and 20days post-fracture (dpf). It also predicted the ratio of cartilage which had become hypertrophic at 10dpf. Following this, three models of fracture healing with increasing levels of angiogenic impairment were developed. Under mild impairment, the model predicted experimentally observed reductions in hypertrophic cartilage at 10dpf as well as the persistence of cartilage at 20dpf. Models of more severe impairment predicted apoptosis and the development of fibrous tissue. These results provide insight into how factors specific to an ischemic callus regulate tissue regeneration and provide support for the hypothesis that chondrocyte hypertrophy and endochondral ossification during tissue regeneration are inhibited by low oxygen.

Gata2 Is a Rheostat for Mesenchymal Stem Cell Fate in Male Mice.

Gata2 is a zinc finger transcription factor that is important in hematopoiesis and neuronal development. However, the roles of Gata2 in the mesenchymal lineages are poorly understood. In vitro studies suggest that Gata2 modulates adipocyte differentiation and mesenchymal stem cell (MSC) proliferation. To systematically determine the in vivo functions of Gata2 in the MSC lineage commitment and development, we have generated three mouse models in which Gata2 is specifically deleted in MSCs, adipocytes, or osteoblasts. During the MSC expansion stage, Gata2 promotes proliferation and attenuates differentiation; thereby Gata2 loss in MSCs results in enhanced differentiation of both adipocytes and osteoblasts. During the differentiation stage, Gata2 also plays MSC-independent roles to impede lineage commitment; hence, Gata2 loss in adipocyte or osteoblast lineages also augments adipogenesis and osteoblastogenesis, respectively. These findings reveal Gata2 as a crucial rheostat of MSC fate to control osteoblast and adipocyte lineage development.

Nanosheet-pore topographical titanium substrates: a biophysical regulator of the fate of mesenchymal stem cells.

Recent reports have demonstrated that nano- or micro-scale topography could enhance the cellular functions of stem cells. In this study, a sub-micrometer topography composed of nanosheet-pore structures was fabricated on the pure titanium surface by a simple vapor alkaline-treatment method to understand more profoundly sub-micrometer topography mediated stem cell behaviors. The topography was characterized by scanning electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy, X-ray diffraction and contact angle measurements, respectively. It specifically mediated cellular functions of rat bone marrow-derived mesenchymal stem cells (MSCs) on cellular and molecular levels under either normal medium or osteoinductive medium conditions. The experimental results indicated that the topography dramatically promoted the adhesion of MSCs grown on the surface, but the shape, morphology and spreading of cells were not significantly affected. In addition, the study demonstrated that the formation of focal adhesion complexes (FAs) were highly dependent on the topography, which in turn affects the subsequent biological functions of MSCs, especially accelerating osteogenic differentiation of MSCs under different conditions. Overall, the sub-micrometer topographical titanium substrate was an excellent biophysical regulator of the fate of mesenchymal stem cells, specifically inducing their differentiation into osteoblasts.

Fate and Effect of Intravenously Infused Mesenchymal Stem Cells in a Mouse Model of Hepatic Ischemia Reperfusion Injury and Resection.

Liver ischemia reperfusion injury (IRI) is inevitable during transplantation and resection and is characterized by hepatocellular injury. Therapeutic strategies to reduce IRI and accelerate regeneration could offer major benefits. Mesenchymal stem cells (MSC) are reported to have anti-inflammatory and regeneration promoting properties. We investigated the effect of MSC in a model of combined IRI and partial resection in the mouse. Hepatic IRI was induced by occlusion of 70% of the blood flow during 60 minutes, followed by 30% hepatectomy. 2 × 105 MSC or PBS were infused 2 hours before or 1 hour after IRI. Six, 48, and 120 hours postoperatively mice were sacrificed. Liver damage was evaluated by liver enzymes, histology, and inflammatory markers. Regeneration was determined by liver/body weight ratio, proliferating hepatocytes, and TGF-β levels. Fate of MSC was visualized with 3D cryoimaging. Infusion of 2 × 105 MSC 2 hours before or 1 hour after IRI and resection showed no beneficial effects. Tracking revealed that MSC were trapped in the lungs and did not migrate to the site of injury and many cells had already disappeared 2 hours after infusion. Based on these findings we conclude that intravenously infused MSC disappear rapidly and were unable to induce beneficial effects in a clinically relevant model of IRI and resection.

Influence of strontium ions incorporated into nanosheet-pore topographical titanium substrates on osteogenic differentiation of mesenchymal stem cells in vitro and on osseointegration in vivo.

Biophysical cues or biochemical cues were proved to efficiently regulate the fate of mesenchymal stem cells (MSCs), but their synergistic effects on the biological functions of MSCs remain to be further investigated. In this study, titanium (Ti) substrates were fabricated with distinct sub-micrometer nanosheet-pore topography via a vapor alkaline treatment method. Strontium (Sr) ions were then incorporated into the Ti substrates via ion exchange. Apart from the influence of biophysical cues from topography, MSCs were simultaneously affected by the biochemical cues from the continuously released Sr ions. The MSCs grown onto Ti substrates with Sr incorporated in them displayed higher (p < 0.05 or p < 0.01) cellular functions than those of pure Ti substrates, including proliferation, the genes and proteins expressions of osteogenic markers and mineralization potential when comparing them with the results of those MSCs grown onto pure Ti substrates. Furthermore, the in vivo investigations demonstrated that the Sr incorporated Ti implants promoted new bone formation. All the results indicated that the incorporated Sr ions and the nanosheet-pore topography of the Ti substrates synergistically enhanced the osteogenic differentiation of MSCs in vitro and osseointegration in vivo. This study advances the understanding of the synergistic influence of biophysical cues and biochemical cues on MSC osteogenic differentiation.

Directed Differentiation of Human-Induced Pluripotent Stem Cells to Mesenchymal Stem Cells.

Multipotent stromal cells, also known as mesenchymal stem cells (MSCs), possess great potential to generate a wide range of cell types including endothelial cells, smooth muscle cells, bone, cartilage, and lipid cells. This protocol describes in detail how to perform highly efficient, lineage-specific differentiation of human-induced pluripotent stem cells (iPSCs) with an MSCs fate. The approach uses a clinically compliant protocol with chemically defined media, feeder-free conditions, and a CD105 positive and CD24 negative selection to achieve a single cell-based MSCs derivation from differentiating human pluripotent cells in approximately 20 days. Cells generated with this protocol express typical MSCs surface markers and undergo adipogenesis, osteogenesis, and chondrogenesis similar to adult bone marrow-derived MSCs (BM-MSCs). Nonetheless, compared with adult BM-MSCs, iPSC-MSCs display a higher proliferative capacity, up to 120 passages, without obvious loss of self-renewal potential and constitutively express MSCs surface antigens. MSCs generated with this protocol have numerous applications, including expansion to large scale cell numbers for tissue engineering and the development of cellular therapeutics. This approach has been used to rescue limb ischemia, allergic disorders, and cigarette smoke-induced lung damage and to model mesenchymal and vascular disorders of Hutchinson-Gilford progeria syndrome (HGPS).

Cell Fate and Differentiation of Bone Marrow Mesenchymal Stem Cells.

Osteoblasts and bone marrow adipocytes originate from bone marrow mesenchymal stem cells (BMMSCs) and there appears to be a reciprocal relationship between adipogenesis and osteoblastogenesis. Alterations in the balance between adipogenesis and osteoblastogenesis in BMMSCs wherein adipogenesis is increased relative to osteoblastogenesis are associated with decreased bone quality and quantity. Several proteins have been reported to regulate this reciprocal relationship but the exact nature of the signals regulating the balance between osteoblast and adipocyte formation within the bone marrow space remains to be determined. In this review, we focus on the role of Transducin-Like Enhancer of Split 3 (TLE3), which was recently reported to regulate the balance between osteoblast and adipocyte formation from BMMSCs. We also discuss evidence implicating canonical Wnt signalling, which plays important roles in both adipogenesis and osteoblastogenesis, in regulating TLE3 expression. Currently, there is demand for new effective therapies that target the stimulation of osteoblast differentiation to enhance bone formation. We speculate that reducing TLE3 expression or activity in BMMSCs could be a useful approach towards increasing osteoblast numbers and reducing adipogenesis in the bone marrow environment.

Polymer micropatterning induces spatially organised fibronectin nanonetworks for efficient integrin/growth factor interaction

Event Abstract Back to Event Polymer micropatterning induces spatially organised fibronectin nanonetworks for efficient integrin/growth factor interaction Annie Zhe Cheng1, 2, Nikolaj Gadegaard1, Matthew Dalby2* and Manuel Salmeron-Sanchez1* 1 University of Glasgow, Biomedical Engineering, United Kingdom 2 University of Glasgow, Centre for Cell Engineering, United Kingdom Introduction: The study of material-protein interactions is important in the development of biomaterials for tissue engineering. We previously showed that poly(ethyl acrylate) (PEA) induces the organisation of fibronectin (FN) into nanonetworks in a process known as material-driven fibrillogenesis[1] and exposes specific structural domains of FN for growth factor (GF) binding[2]. In addition, the incorporation of microscale features is interesting in biomaterials as it allows the mimicking of in vivo systems, whereby cells thrive in a microscale environment[3]. Here we use a simple photolithographic procedure to fabricate micropatterns of PEA, allowing material-driven fibrillogenesis of FN to occur in microscale regions. The synergistic relationship between FN nanonetworks and growth factors is also investigated with respect to mesenchymal stem cell (MSC) response, in particular adhesion, migration, and differentiation. Materials and Methods: Photolithography (PL): PEA is spin-coated onto glass substrates, which are subsequently coated with photoresist and patterned with a photomask via exposure to UV light. Oxygen plasma is used to etch PEA and expose glass for PEG-silane grafting, yielding a non-fouling background. Residual photoresist is removed to expose the underlying PEA for protein coating. Surface characterisation: Micropatterned surfaces coated with FN and BMP-2 on PEA are characterised by SEM, AFM, immunogold staining, and immunofluorescence to verify successfu protein patterning on the substrates. Cell culture: MSCs are seeded on micropatterned, PEA-modified surfaces coated with FN and/or BMP-2 for up to 4 weeks to study adhesion, migration, and differentiation. Results and Discussion: PEA is patterned on 100 μm × 100 μm square islands using PL. FN is coated onto these patterns and immunostaining of FN is carried out to visualise the resulting FN microdomains. The interaction between FN and PEA is confirmed using AFM, which reveals FN nanonetworks on the microdomains. Additionally, BMP-2 localisation is visualised by immunogold labelling. When MSCs are cultured on micropatterned, protein-modified surfaces, they selectively adhere to the FN microdomains while avoiding the non-fouling background. The osteogenic potential of these micropatterned surfaces is investigated. With the incorporation of BMP-2 within the FN nanonetworks, MSC differentiation into osteoblasts is observed on the microscale islands, indicating the ability to spatially control MSC differentiation using bioactive cues including chemical interfaces and GF signalling. Conclusions: We demonstrate a simple method of patterning microdomains of PEA using PL and subsequently coating the microdomains with FN and BMP-2 to facilitate synergistic interactions and influence MSC fate. This approach can be utilized with different shapes, dimensions, and order, and can also be downsized to yield much smaller feature sizes, enabling the precise design of bioactive surfaces to promote osteogenesis in MSCs. Medical Research Council; James Watt Nanofabrication Centre

Role of oxygen as a regulator of stem cell fate during the spontaneous repair of osteochondral defects

The complexity of the in vivo environment makes it is difficult to isolate the effects of specific cues on regulating cell fate during regenerative events such as osteochondral defect repair. The objective of this study was to develop a computational model to explore how joint specific environmental factors regulate mesenchymal stem cell (MSC) fate during osteochondral defect repair. To this end, the spontaneous repair process within an osteochondral defect was simulated using a tissue differentiation algorithm which assumed that MSC fate was regulated by local oxygen levels and substrate stiffness. The developed model was able to predict the main stages of tissue formation observed by a number of in vivo studies. Following this, a parametric study was conducted to better understand why interventions that modulate angiogenesis dramatically impact the outcome of osteochondral defect healing. In the simulations where angiogenesis was reduced, by week 12, the subchondral plate was predicted to remain below the native tidemark, although the chondral region was composed entirely of cartilage and fibrous tissue. In the simulations where angiogenesis was increased, more robust cell proliferation and cartilage formation were observed during the first 4 weeks, however, by week 12 the subchondral plate had advanced above the native tidemark although any remaining tissue was either hypertrophic cartilage or fibrous tissue. These results suggest that osteochondral defect repair could be enhanced by interventions where angiogenesis is promoted but confined to within the subchondral region of the defect. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1026-1036, 2016.

The Fate and Distribution of Autologous Bone Marrow Mesenchymal Stem Cells with Intra-Arterial Infusion in Osteonecrosis of the Femoral Head in Dogs.

This study aimed to investigate if autologous bone marrow mesenchymal stem cells (MSCs) could treat osteonecrosis of the femoral head (ONFH) and what the fate and distribution of the cells are in dogs. Twelve Beagle dogs were randomly divided into two groups: MSCs group and SHAM operated group. After three weeks, dogs in MSCs group and SHAM operated group were intra-arterially injected with autologous MSCs and 0.9% normal saline, respectively. Eight weeks after treatment, the necrotic volume of the femoral heads was significantly reduced in MSCs group. Moreover, the trabecular bone volume was increased and the empty lacunae rate was decreased in MSCs group. In addition, the BrdU-positive MSCs were unevenly distributed in femoral heads and various vital organs. But no obvious abnormalities were observed. Furthermore, most of BrdU-positive MSCs in necrotic region expressed osteocalcin in MSCs group and a few expressed peroxisome proliferator-activated receptor-γ (PPAR-γ). Taken together, these data indicated that intra-arterially infused MSCs could migrate into the necrotic field of femoral heads and differentiate into osteoblasts, thus improving the necrosis of femoral heads. It suggests that intra-arterial infusion of autologous MSCs might be a feasible and relatively safe method for the treatment of femoral head necrosis.

The Role of Bone Marrow Microenvironment in Governing the Balance between Osteoblastogenesis and Adipogenesis.

In the adult bone marrow, osteoblasts and adipocytes share a common precursor called mesenchymal stem cells (MSCs). The plasticity between the two lineages has been confirmed over the past decades, and has important implications in the etiology of bone diseases such as osteoporosis, which involves an imbalance between osteoblasts and adipocytes. The commitment and differentiation of bone marrow (BM) MSCs is tightly controlled by the local environment that maintains a balance between osteoblast lineage and adipocyte. However, pathological conditions linked to osteoporosis can change the BM microenvironment and shift the MSC fate to favor adipocytes over osteoblasts, and consequently decrease bone mass with marrow fat accumulation. This review discusses the changes that occur in the BM microenvironment under pathological conditions, and how these changes affect MSC fate. We suggest that manipulating local environments could have therapeutic implications to avoid bone loss in diseases like osteoporosis.

Nascent osteoblast matrix inhibits osteogenesis of human mesenchymal stem cells in vitro.

IntroductionAdult mesenchymal stem cells (MSCs) are considered promising candidates for cell-based therapies. Their potential utility derives primarily from their immunomodulatory activity, multi-lineage differentiation potential, and likely progenitor cell function in wound healing and repair of connective tissues. However, in vitro, MSCs often senesce and spontaneously differentiate into osteoblasts after prolonged expansion, likely because of lack of regulatory microenvironmental signals. In vivo, osteoblasts that line the endosteal bone marrow surface are in close proximity to MSCs in the marrow stroma and thus may help to regulate MSC fate.MethodsWe examined here how osteogenic differentiation of MSCs in vitro is affected by exposure to osteoblastic cells (OBCs). Human bone marrow MSCs were exposed to OBCs, derived by induced osteogenic differentiation of MSCs, either directly in contact co-cultures, or indirectly to OBC-conditioned medium or decellularized OBC extracellular matrix (ECM).ResultsOur results showed that OBCs can act as negative regulators of MSC osteogenesis. mRNA expression profiling revealed that OBCs did not affect MSC osteogenesis in direct contact cultures or via secreted factors. However, seeding MSCs on decellularized OBC ECM significantly decreased expression of several osteogenic genes and maintained their fibroblastic morphologies. Proteomic analysis identified some of the candidate protein regulators of MSC osteogenesis.ConclusionsThese findings provide the basis for future studies to elucidate the signaling mechanisms responsible for osteoblast matrix-mediated regulation of MSC osteogenesis and to better manipulate MSC fate in vitro to minimize their spontaneous differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0223-x) contains supplementary material, which is available to authorized users.

Transforming Growth Factor-β-Induced KDM4B Promotes Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

The high prevalence of cartilage diseases and limited treatment options create a significant biomedical burden. Due to the inability of cartilage to regenerate itself, introducing chondrocyte progenitor cells to the affected site is of significant interest in cartilage regenerative therapies. Tissue engineering approaches using human mesenchymal stem cells (MSCs) are promising due to their chondrogenic potential, but a comprehensive understanding of the mechanisms governing the fate of MSCs is required for precise therapeutic applications in cartilage regeneration. TGF-β is known to induce chondrogenesis by activating SMAD signaling pathway and upregulating chondrogenic genes such as SOX9; however, the epigenetic regulation of TGF-β-mediated chondrogenesis is not understood. In this report, we found that TGF-β dramatically induced the expression of KDM4B in MSCs. When KDM4B was overexpressed, chondrogenic differentiation was significantly enhanced while KDM4B depletion by shRNA led to a significant reduction in chondrogenic potential. Mechanistically, upon TGF-β stimulation, KDM4B was recruited to the SOX9 promoter, removed the silencing H3K9me3 marks, and activated the transcription of SOX9. Furthermore, KDM4B depletion reduced the occupancy of SMAD3 in the SOX9 promoter, suggesting that KDM4B is required for SMAD-dependent coactivation of SOX9. Our results demonstrate the critical role of KDM4B in the epigenetic regulation of TGF-β-mediated chondrogenic differentiation of MSCs. Since histone demethylases are chemically modifiable, KDM4B may be a novel therapeutic target in cartilage regenerative therapy.

SIRT1 inhibits adipogenesis and promotes myogenic differentiation in C3H10T1/2 pluripotent cells by regulating Wnt signaling.

BackgroundThe directed differentiation of mesenchymal stem cells (MSCs) is tightly controlled by a complex network. Wnt signaling pathways have an important function in controlling the fate of MSCs. However, the mechanism through which Wnt/β-catenin signaling is regulated in differentiation of MSCs remains unknown. SIRT1 plays an important role in the regulation of MSCs differentiation.ResultsThis study aimed to determine the effect of sirtuin 1 (SIRT1) on adipogenesis and myogenic differentiation of C3H10T1/2 cells. First, the MSC commitment and differentiation model was established by using 5-azacytidine. Using the established model, C3H10T1/2 cells were treated with SIRT1 activator/inhibitor during differentiation. The results showed that resveratrol inhibits adipogenic differentiation and improves myogenic differentiation, whereas nicotinamide promotes adipogenic differentiation. Notably, during commitment, resveratrol blocked adipocyte formation and promoted myotubes differentiation, whereas nicotinamide enhanced adipogenic potential of C3H10T1/2 cells. Furthermore, resveratrol elevated the expression of Cyclin D1 and β-catenin in the early stages. The luciferase assay showed that knockdown SIRT1 inhibits Wnt/β-catenin signaling, while resveratrol treatment or overexpression SIRT1 activates Wnt/β-catenin signaling. SIRT1 suppressed the expression of Wnt signaling antagonists sFRP2 and DACT1. Knockdown SIRT1 promoted adipogenic potential of C3H10T1/2 cells, whereas overexpression SIRT1 inhibited adipogenic differentiation and promoted myogenic differentiation.ConclusionsTogether, our results suggested that SIRT1 inhibits adipogenesis and stimulates myogenic differentiation by activating Wnt signaling.

Abstract 13828: Fibroblast Growth Factor-23 Induces Cellular Senescence in Human Mesenchymal Stem Cells From Skeletal Muscle Independently of Klotho Pathway

Introduction: Mesenchymal stem cells (MSCs) in skeletal muscle represent a distinct cell population from satellite cells (StCs), and the functional impairment have shown to be associated with ectopic fat formation in muscle tissues. Muscle wasting and/or degeneration are prevalent in heart failure and chronic kidney disease. However, it remains unknown whether such disease condition influences the MSC function and fate. Thus, aim of this study was to investigate effects of fibroblast growth factor (FGF)-23 and angiotensin (ANG)-II, neurohumoral factors related to pathophysiology of cardio-renal syndrome (CRS), on the MSCs in culture system. Methods and Results: MSCs were isolated from human skeletal muscle and cultured with appropriate growth medium. Gene expression analysis demonstrated the enhanced expression of FGF receptors and ANG-II type 1 receptor in the MSCs as compared with the human StCs. Klotho gene was not detected in the two progenitors. The cultured MSCs and StCs were treated with FGF-23 (10ng/ml) and ANG-II (100 nM) for 48 hours. Cell proliferation assay revealed significant decreased cell number in the MSCs treated with FGF-23 but not ANG-II than in the controls (58 % of the controls, p&lt; 0.05). The neurohumoral factors did not affect the cell counts of the StCs. The FGF-23 treated MSCs exhibited the senescent phenotype, as judged by senescence-associated β-galactosidase assay (%senescent cells: 46.5± 11.7 % in the FGF-23 vs. 35.8± 5.8% in the controls, p&lt; 0.05), cell morphology, and increased expression of p53 and p21 in western blot analysis. FGF-23 also augmented intracellular oxidative stress in the MSCs. The mechanisms responsible for the accumulation of reactive oxygen species (ROS) were attributable to increased NADPH oxidase and decreased MnSOD expression in the cells. In histological study, p53-positive senescent MSCs were detected in adipose infiltration area of degenerated gastrocunemius muscle tissues from a CRS patient. Conclusions: Results of this study indicate that FGF-23 induces premature senescence in human MSCs from skeletal muscle via promoting ROS/p53/p21 pathway in klotho-independent action. Interaction of the MSCs with FGF-23 may play a key role in muscle wasting in patients with CRS.

The fate of systemically administrated allogeneic mesenchymal stem cells in mouse femoral fracture healing.

IntroductionThe fate and whereabouts of the allogenic mesenchymal stem cells (MSCs) following their transplantation are not well understood. The present study investigated the fate of systemically administrated allogeneic MSCs in mouse fracture healing by using in vivo imaging and immunohistochemistry methods.MethodsOpen femoral fracture with internal fixation was established in 30 FVB mice, which were assigned to three groups receiving phosphate-buffered saline (PBS) injection, MSC systemic injection, or MSC local injection. Luc-MSCs (5 × 105) isolated from the luciferase transgenic mice with FVB background were injected at 4 days after fracture. All animals were terminated at 5 weeks after fracture; examinations included bioluminescence-based in vivo imaging, micro-computer tomography, mechanical testing, histology, immunohistochemistry, and double immunofluorescence staining.ResultsThe bioluminescence signals of the Luc-MSCs at the fracture site could be detected for 12–14 days following their injection in the Luc-MSC local injection group, whereas in the Luc-MSC systemic injection group, Luc-MSCs were initially trapped in lungs for about 8–9 days and then gradually redistributed to the fracture site. Bone mineral density, bone volume/tissue volume, ultimate load, and E-modulus in the MSC injection groups were significantly higher than those in the PBS group. Double immunostaining demonstrated that the MSC local injection group had more Luc-positive cells, and there was a higher apoptotic rate at the fracture site than the MSC systemic injection group. Both Luciferase-positive MSCs and osteoblasts were present in the callus in the MSC injection groups at 5 weeks after fracture, suggesting that some of allogenic Luc-MSCs contributed to the new bone formation. Only less than 3 % of injected Luc-MSCs remained at the fracture site in the MSC injection groups at 5 weeks following the fracture, and the rest of the injected Luc-MSCs disappeared.ConclusionsOur data showed that both systemic and local injection of allogeneic MSCs promoted fracture healing through enhancing biomechanical properties, bone content, and enlarged callus sizes. Immunohistochemistry confirmed that the injected MSCs are still present in the fracture site and can differentiate into osteoblasts to participate in fracture healing even at 5 weeks following the fracture. These findings provide useful information for the use of allogenic MSCs for cell therapy applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0198-7) contains supplementary material, which is available to authorized users.

HOTAIR and its surrogate DNA methylation signature indicate carboplatin resistance in ovarian cancer.

BackgroundUnderstanding carboplatin resistance in ovarian cancer is critical for the improvement of patients’ lives. Multipotent mesenchymal stem cells or an aggravated epithelial to mesenchymal transition phenotype of a cancer are integrally involved in pathways conferring chemo-resistance. Long non-coding RNA HOTAIR (HOX transcript antisense intergenic RNA) is involved in mesenchymal stem cell fate and cancer biology.MethodsWe analyzed HOTAIR expression and associated surrogate DNA methylation (DNAme) in 134 primary ovarian cancer cases (63 received carboplatin, 55 received cisplatin and 16 no chemotherapy). We validated our findings by HOTAIR expression and DNAme analysis in a multicentre setting of five additional sets, encompassing 946 ovarian cancers. Chemo-sensitivity has been assessed in cell culture experiments.ResultsHOTAIR expression was significantly associated with poor survival in carboplatin-treated patients with adjusted hazard ratios for death of 3.64 (95 % confidence interval [CI] 1.78–7.42; P < 0.001) in the discovery and 1.63 (95 % CI 1.04–2.56; P = 0.032) in the validation set. This effect was not seen in patients who did not receive carboplatin (0.97 [95 % CI 0.52–1.80; P = 0.932]). HOTAIR expression or its surrogate DNAme signature predicted poor outcome in all additional sets of carboplatin-treated ovarian cancer patients while HOTAIR expressors responded preferentially to cisplatin (multivariate interaction P = 0.008).ConclusionsNon-coding RNA HOTAIR or its more stable DNAme surrogate may indicate the presence of a subset of cells which confer resistance to carboplatin and can serve as (1) a marker to personalise treatment and (2) a novel target to overcome carboplatin resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0233-4) contains supplementary material, which is available to authorized users.

A combinatorial variation in surface chemistry and pore size of three-dimensional porous poly(ε-caprolactone) scaffolds modulates the behaviors of mesenchymal stem cells

Biomaterial properties play significant roles in controlling cellular behaviors. The objective of the present study was to investigate how pore size and surface chemistry of three-dimensional (3D) porous scaffolds regulate the fate of mesenchymal stem cells (MSCs) in vitro in combination. First, on poly(ε-caprolactone) (PCL) films, the hydrolytic treatment was found to stimulate the adhesion, spreading and proliferation of human MSCs (hMSCs) in comparison with pristine films, while the aminolysis showed mixed effects. Then, 3D porous PCL scaffolds with varying pore sizes (100–200μm, 200–300μm and 300–450μm) were fabricated and subjected to either hydrolysis or aminolysis. It was found that a pore size of 200–300μm with hydrolysis in 3D scaffolds was the most favorable condition for growth of hMSCs. Importantly, while a pore size of 200–300μm with hydrolysis for 1h supported the best osteogenic differentiation of hMSCs, the chondrogenic differentiation was greatest in scaffolds with a pore size of 300–450μm and treated with aminolysis for 1h. Taken together, these results suggest that surface chemistry and pore size of 3D porous scaffolds may potentially have a synergistic impact on the behaviors of MSCs.

Chondrogenic Differentiation of Human Mesenchymal Stem Cells Results in Substantial Changes of Ecto-Nucleotides Metabolism.

Mesenchymal stem cells (MSCs) are population of adult stem cells and attractive candidates for cartilage repair due to their chondrogenic potential. Purinergic compounds (purinergic receptors and ecto-enzymes metabolizing nucleotides), together with nucleotides/nucleosides present in the extracellular environment, are known to play a key role in controlling the stem cells biological potential to proliferate and differentiate. Despite the available literature pointing to the importance of purinergic signaling in controlling the fate of MSCs, the research results linking nucleotides and ecto-nucleotidases with MSCs chondrogenic differentiation are indigent. Therefore, the aim of presented study was the characterization of the ecto-nucleotides hydrolysis profile and ecto-enzymes expression in human umbilical cord-derived MSCs and chondrogenically induced MSCs. We described substantial changes of ecto-nucleotides metabolism and ecto-enzymes expression profiles resulting from chondrogenic differentiation of human umbilical cord-derived MSCs. The increased rate of ADP hydrolysis, measured by ecto-nucleotidases activity, plays a pivotal role in the regulation of cartilage formation and resorption. Despite the increased level of NTPDase1 and NTPDase3 mRNA expression in chondrogenically induced MSCs, their activity toward ATP remains quite low. Supported by the literature data, we hypothesize that structure-function relationships in chondrogenic lineage dictate the direction of nucleotides metabolism. In early neocartilage tissue, the beneficial role of ATP in improving biomechanical properties of cartilage does not necessitate the high rate of enzymatic ATP degradation.

Substrate Coupling Strength of Integrin-Binding Ligands Modulates Adhesion, Spreading, and Differentiation of Human Mesenchymal Stem Cells.

Substrate stiffness has been shown to regulate the differentiation fate of human mesenchymal stem cells (hMSCs). hMSCs sense and respond to substrate rigidity by exerting traction forces upon the binding between integrins and integrin-specific ligands present on the substrate surface. However, in previous studies, integrin-specific ligands such as Arg-Gly-Asp (RGD) peptides are always grafted to the substrate by a permanent covalent bond. Whether the coupling strength of integrin-specific ligands on substrate will influence cell behaviors has not been explored. In this work, we have developed a facile platform to investigate the effects of varied coupling strength between the RGD peptide and the glass substrate on stem cell behaviors. Glass coverslips are decorated with positive charges by silanization using (3-aminopropyl) triethoxysilane (APTES) to immobilize negatively charged citrate-capped gold nanoparticles (cit-AuNPs) solely via electrostatic interactions. The monolayer of electrostatically immobilized cit-AuNPs is further conjugated with the thiolated RGD peptides through the sulfur-gold bond. The substrate coupling strength of the RGD peptides, which is dependent on the electrostatic interactions between the APTES-treated glass substrate and the cit-AuNPs, is simply tuned by changing the APTES dosage and, hence, the resultant positive charge density on the surface. A total of 0.5% and 12.5% of APTES are used to fabricate low-coupling-strength surfaces (namely, LCS0.5 and LCS12.5), whereas 25% and 50% of APTES are used to fabricate high-coupling-strength surfaces (namely, HCS25 and HCS50). Fluorescence microscopy shows that hMSCs spread well and form stable actin filamentous structure on HCS surfaces but not on LCS surfaces. Remarkably, hMSCs exhibit enhanced osteogenesis on HCS surfaces as revealed by the immunostaining results of multiple early osteogenic markers. These differential behaviors may be governed by Yes-associated protein (YAP), a mechanosensitive transcriptional regulator of stem cells. Our findings highlight the importance of the substrate coupling strength of integrin-binding ligands on regulating adhesion, spreading, and differentiation of hMSCs.