Human neural stem cell-derived cultures in three- dimensional substrates form spontaneously functional neuronal networks

Differentiated human neural stem cells were cultured in an inert three-dimensional (3D) scaffold and, unlike two-dimensional (2D) but otherwise comparable monolayer cultures, formed spontaneously active, functional neuronal networks that responded reproducibly and predictably to conventional pharmacological treatments to reveal functional, glutamatergic synapses. Immunocytochemical and electron microscopy analysis revealed a neuronal and glial population, where markers of neuronal maturity were observed in the former. Oligonucleotide microarray analysis revealed substantial differences in gene expression conferred by culturing in a 3D vs a 2D environment. Notable and numerous differences were seen in genes coding for neuronal function, the extracellular matrix and cytoskeleton. In addition to producing functional networks, differentiated human neural stem cells grown in inert scaffolds offer several significant advantages over conventional 2D monolayers. These advantages include cost savings and improved physiological relevance, which make them better suited for use in the pharmacological and toxicological assays required for development of stem cell-based treatments and the reduction of animal use in medical research.

Translational research and therapeutic applications of neural crest-derived stem cells in regenerative periodontology

Regeneration of periodontal tissues aims to utilize tissue engineering techniques to restore lost periodontal tissues including the cementum, periodontal ligament and alveolar bone. Regenerative dentistry and its special field regenerative periodontology represent relatively new and emerging branches of translational stem cell biology and regenerative medicine focusing on replacing and regenerating dental tissues to restore or re-establish their normal function lost during degenerative diseases or acute lesions. The regeneration itself can be achieved through transplantation of autologous or allogenic stem cells, or by improving the tissue self-repair mechanisms (e.g. by application of growth factors). In addition, a combination of stem cells or stem cell-containing tissue with bone implants can be used to improve tissue integration and the clinical outcome. As the oral cavity represents a complex system consisting of teeth, bone, soft tissues and sensory nerves, regenerative periodontology relies on the use of stem cells with relatively high developmental potential. Notably, the potential use of pluripotent stem cell types such as human embryonic stem cells or induced pluripotent stem cells is still aggravated by ethical and practical problems. Thus, other cellular sources such as those readily available in the postnatal craniofacial area and particularly in oral structures offer a much better and realistic alternative as cellular regenerative sources. In this review, we summarize current knowledge on the oral neural crest-derived stem cell populations (oNCSCs) and discuss their potential in regenerative periodontology.

Systemic delivery of adult stem cells improves cardiac function in spontaneously hypertensive rats

Heart regeneration after myocardial infarction (MI) can occur after cell therapy, but the mechanisms, cell types and delivery methods responsible for this improvement are still under investigation. In the present study, we evaluated the impact of systemic delivery of bone marrow cells (BMC) and cultivated mesenchymal stem cells (MSC) on cardiac morphology, function and mortality in spontaneously hypertensive rats (SHR) submitted to coronary occlusion. Female syngeneic adult SHR, submitted or not (control group; C) to MI, were treated with intravenous injection of MSC (MI + MSC) or BMC (MI + BM) from male rats and evaluated after 1, 15 and 30 days by echocardiography. Systolic blood pressure (SBP), functional capacity, histology, mortality rate and polymerase chain reaction for the Y chromosome were also analysed. Myocardial infarction induced a decrease in SBP and BMC, but not MSC, prevented this decrease. An improvement in functional capacity and ejection fraction (38 +/- 4, 39 +/- 3 and 58 +/- 2% for MI, MI + MSC and MI + BM, respectively; P < 0.05), as well as a reduction of the left ventricle infarcted area, were observed in rats from the MI + BM group compared with the other three groups. Treated animals had a significantly reduced lesion tissue score. The mortality rate in the C, MI + BM, MI + MSC and MI groups was 0, 0, 16.7 and 44.4%, respectively (P < 0.05 for the MI + MSC and MI groups compared with the C and MI + BM groups). The results of the present study suggest that systemic administration of BMC can improve left ventricular function, functional capacity and, consequently, reduce mortality in an animal model of MI associated with hypertension. We speculate that the cells transiently home to the myocardium, releasing paracrine factors that recruit host cells to repair the lesion.

Isolation and characterization of Wharton's jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: An improved approach in cartilage tissue engineering

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n=5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1×105) were than encapsulated inside 60μl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI. © 2013 Informa UK Ltd.

Isolation and characterization of equine peripheral blood-derived multipotent mesenchymal stromal cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of mesenchymal stem cell migration after equine tendonitis therapy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Clopidogrel enhances mesenchymal stem cell proliferation following periodontitis

Bone formation is dependent on the differentiation of osteoblasts from mesenchymal stem cells (MSCs). In addition to serving as progenitors, MSCs reduce inflammation and produce factors that stimulate tissue formation. Upon injury, MSCs migrate to the periodontium, where they contribute to regeneration. We examined the effect of clopidogrel and aspirin on MSCs following induction of periodontitis in rats by placement of ligatures. We showed that after the removal of ligatures, which induces resolution of periodontal inflammation, clopidogrel had a significant effect on reducing the inflammatory infiltrate. It also increased the number of osteoblasts and MSCs. Mechanistically, the latter was linked to increased proliferation of MSCs in vivo and in vitro. When given prior to inducing periodontitis, clopidogrel had little effect on MSC or osteoblasts numbers. Applying aspirin before or after induction of periodontitis did not have a significant effect on the parameters measured. These results suggest that clopidogrel may have a positive effect on MSCs in conditions where a reparative process has been initiated.

Mesenchymal stem cell enhances chondral defects healing in horses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

Lima S.A.F., Wodewotzky T.I., Lima-Neto J.F., Beltrao-Braga P.C.B. & Alvarenga F.C.L. 2012. [In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors.] Diferenciacao in vitro de celulas-tronco mesenquimais da medula ossea de caes em precursores osteogenicos. Pesquisa Veterinaria Brasileira 32(5):463-469. Departamento de Reproducao Animal e Radiologia Veterinaria, Faculdade de Medicina Veterinaria e Zootecnia, Universidade Estadual Paulista, Campus de Botucatu, Distrito de Rubiao Junior s/n, Botucatu, SP 18618-970, Brazil. E-mail: silviavet@usp.br The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12. Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.

Lung on a chip: Co-culture of alveolar epithelial cells and bone marrow derived stromal stem cells in a microfluidic system

Background: Microfluidics system are novel tools to study cell-cell interactions in vitro. This project focuses on the development of a new microfluidic device to co-culture alveolar epithelial cells and mesenchymal stem cells to study cellular interactions involved in healing the injured alveolar epithelium. Methods: Microfluidic systems in polydimethylsiloxane were fabricated by soft lithography. The alveolar A549 epithelial cells were seeded and injury tests were made on the cells by perfusion with media containing H2O2 or bleomycin during 6 or 18hrs. Rat Bone marrow derived stromal cells (BMSC) were then introduced into the system and cell-cell interaction was studied over 24 hrs. Results: A successful co-culture of A549 alveolar epithelial cells and BMS was achieved in the microfluidic system. The seeded alveolar epithelial cells and BMSC adhered to the bottom surface of the microfluidic device and proliferated under constant perfusion. Epithelial injury to mimic mechanisms seen in idiopathic pulmonary fibrosis was induced in the microchannels by perfusing with H2O2 or bleomycin. Migration of BMSC towards the injured epithelium was observed as well as cell-cell interaction between the two cell types was also seen. Conclusion: We demonstrate a novel microfluidic device aimed at showing interactions between different cell types on the basis of a changing microenvironment. Also we were able to confirm interaction between injured alvolar epithelium and BMSC, and showed that BMSC try to heal the injured epitelium.

Enhanced early chondrogenesis in articular defects following arthroscopic mesenchymal stem cell implantation in an equine model

Mesenchymal stem cells (MSCs) provide an important source of pluripotent cells for musculoskeletal tissue repair. This study examined the impact of MSC implantation on cartilage healing characteristics in a large animal model. Twelve full-thickness 15-mm cartilage lesions in the femoropatellar articulations of six young mature horses were repaired by injection of a self-polymerizing autogenous fibrin vehicle containing mesenchymal stem cells, or autogenous fibrin alone in control joints. Arthroscopic second look and defect biopsy was obtained at 30 days, and all animals were euthanized 8 months after repair. Cartilage repair tissue and surrounding cartilage were assessed by histology, histochemistry, collagen type I and type II immunohistochemistry, collagen type II in situ hybridization, and matrix biochemical assays. Arthroscopic scores for MSC-implanted defects were significantly improved at the 30-day arthroscopic assessment. Biopsy showed MSC-implanted defects contained increased fibrous tissue with several defects containing predominantly type II collagen. Long-term assessment revealed repair tissue filled grafted and control lesions at 8 months, with no significant difference between stem cell-treated and control defects. Collagen type II and proteoglycan content in MSC-implanted and control defects were similar. Mesenchymal stem cell grafts improved the early healing response, but did not significantly enhance the long-term histologic appearance or biochemical composition of full-thickness cartilage lesions.

FUNCTIONALIZATION OF CARBON NANOTUBES FOR MESENCHYMAL STEM CELL-ASSISTED PHOTOTHERMAL THERAPY

Carbon nanotubes were first cut and functionalized with a newly developed reaction involving autoclaving and sonication in hydrogen peroxide. The functionalized nanotubes were characterized and evaluated for aqueous solubility. Studies which relate reaction conditions to final carbon nanotube length were conducted. Hydroxyl groups present on the carbon nanotubes served as a platform for a series of addition reactions, with the objective of conjugating streptavidin and fluorescent markers onto the carbon nanotubes. The modified nanotubes were attached onto the surface of biotinylated mesenchymal stem cells, creating a novel, tumor-homing delivery system for photothermal anticancer agents.

MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses

OBJECTIVE: MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue-specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA-140 (miR-140). METHODS: To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR-140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin-1beta (IL-1beta) on miR-140 expression. Double-stranded miR-140 (ds-miR-140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA. RESULTS: Microarray analysis showed that miR-140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR-140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR-140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1beta suppressed miR-140 expression. Transfection of chondrocytes with ds-miR-140 down-regulated IL-1beta-induced ADAMTS5 expression and rescued the IL-1beta-dependent repression of AGGRECAN gene expression. CONCLUSION: This study shows that miR-140 has a chondrocyte differentiation-related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1beta may contribute to the abnormal gene expression pattern characteristic of OA.

A papain-induced disc degeneration model for the assessment of thermo-reversible hydrogel-cells therapeutic approach

Nucleus pulposus (NP) regeneration by the application of injectable cell-embedded hydrogels is an appealing approach for tissue engineering. We investigated a thermo-reversible hydrogel (TR-HG), based on a modified polysaccharide with a thermo-reversible polyamide [poly(N-isopropylacrylamide), pNIPAM], which is made to behave as a liquid at room temperature and hardens at > 32 °C. In order to test the hydrogel, a papain-induced bovine caudal disc degeneration model (PDDM), creating a cavity in the NP, was employed. Human mesenchymal stem cells (hMSCs) or autologous bovine NP cells (bNPCs) were seeded in TR-HG; hMSCs were additionally preconditioned with rhGDF-5 for 7 days. Then, TR-HG was reversed to a fluid and the cell suspension injected into the PDDM and kept under static loading for 7 days. Experimental design was: (D1) fresh disc control + PBS injection; (D2) PDDM + PBS injection; (D3) PDDM + TR-HG (material control); (D4) PDDM + TR-HG + bNPCs; (D5) PDDM + TR-HG + hMSCs. Magnetic resonance imaging performed before and after loading, on days 9 and 16, allowed imaging of the hydrogel-filled PDDM and assessment of disc height and volume changes. In gel-injected discs the NP region showed a major drop in volume and disc height during culture under static load. The RT–PCR results of injected hMSCs showed significant upregulation of ACAN, COL2A1, VCAN and SOX9 during culture in the disc cavity, whereas the gene expression profile of NP cells remained unchanged. The cell viability of injected cells (NPCs or hMSCs) was maintained at over 86% in 3D culture and dropped to ~72% after organ culture. Our results underline the need for load-bearing hydrogels that are also cyto-compatible.