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PUBLISHED RESEARCH 2013


A biological therapy to osteoarthritis treatment using platelet-rich plasma
Anitua, Eduardo and S{\'a}nchez, Mikel and Orive, Gorka and Padilla, Sabino
2013 August 23
[Link: informa]

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Abstract:
Introduction:
Osteoarthritis (OA) is a degenerative disease affecting the synovial joint. It is caused by cells exposure to non-physiological stimuli, either mechanical or biochemical, and the loss of bone-cartilage homeostasis. Some of these changes, however, may be reversed by the use of single or combined growth factors, suggesting that the treatment of OA could be addressed using a pool of growth factors.

Areas covered:
This review addresses current molecular and biological knowledge and implicates the recapitulation of some developmental processes during endochondral ossification in OA aetiology and pathogenesis. Platelets act as carriers of endogenous morphogens that may modulate cell fate and therefore affect joint tissues structure and function. We shed light on the platelet-rich plasma effects on biological level that might drive the osteoarthritic joint's improvement both in structure and function.

Expert opinion:
We present the therapeutic potential of plasma rich in growth factors (PRGF-Endoret), an endogenous biological therapy that might modulate the gene expression of cells such as chondrocytes, synoviocytes, macrophages, and mesenchymal stem cells, and thereby influence an anabolic microenvironment of synovial joint which is conducive to maintaining the homeostatic state of the joint's tissues, and hence reduce pain and improve the joint motion.


Unravelling stem cell dynamics by lineage tracing.
Cédric Blanpain & Benjamin D. Simons
2013 July 17
[Link: nature]

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Abstract:
During embryonic and postnatal development, the different cells types that form adult tissues must be generated and specified in a precise temporal manner. During adult life, most tissues undergo constant renewal to maintain homeostasis. Lineage-tracing and genetic labelling technologies are beginning to shed light on the mechanisms and dynamics of stem and progenitor cell fate determination during development, tissue maintenance and repair, as well as their dysregulation in tumour formation. Statistical approaches, based on proliferation assays and clonal fate analyses, provide quantitative insights into cell kinetics and fate behaviour. These are powerful techniques to address new questions and paradigms in transgenic mouse models and other model systems.


Epiregulin can promote proliferation of stem cells from the dental apical papilla via MEK/Erk and JNK signalling pathways
Cao, Y and Xia, D S and Qi, S R and Du, J and Ma, P and Wang, S L and Fan, Z P
2013 July 5
[Link: Wiley]

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Abstract:
Objectives
Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration, but their molecular mechanisms of differentiation and proliferation remain unclear; this situation has restricted use of MSCs to a limited number of applications. A previous study of ours found a member of the epidermal growth factor family, epiregulin (EREG), to be involved in regulation of MSC differentiation. In the present study, we have used human dental stem cells from the apical papilla (SCAPs) to investigate the role of EREG on proliferation of MSCs.

Materials and methods
SCAPs were isolated from apical papillae of immature third molars. Retroviral short hairpin RNA (shRNA) was used to silence EREG gene expression, and human recombinant EREG protein was used to stimulate SCAPs. SCAP proliferation was examined using tetrazolium dye colorimetric assay/cell growth curve. Western blotting was performed to detect expressions of extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), mitogen-activated protein kinases 1 and 2 (MEK1/2), protein kinase B (Akt), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK).

Results
Depletion of EREG with shRNA inhibited SCAP proliferation and repressed phosphorylation of Erk1/2 and JNK. Human recombinant EREG protein promoted cell proliferation and enhanced Erk1/2, MEK and JNK phosphorylation in SCAPs. Furthermore, blocking MEK/Erk signalling with specific Erk1/2 inhibitor PD98059, or JNK signalling with specific inhibitor SP600125, abolished effects of EREG on cell proliferation.

Conclusion
These findings indicate that EREG could enhance cell proliferation in dental tissue-derived MSCs by activating MEK/Erk and JNK signalling pathways.


HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition.
Bonomi, Serena and di Matteo, Anna and Buratti, Emanuele and Cabianca, Daphne S and Baralle, Francisco E and Ghigna, Claudia and Biamonti, Giuseppe
2013 June 10
[Link: Oxford Journals]

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Abstract:
Alternative splicing is a mechanism of gene expression regulation that either modulates the production of protein isoforms with distinct structural and functional properties or affects mRNA stability, through the introduction of premature stop codons, and translatability, by removing targets sites for microRNAs. Its prevalence in regulatory circuits is proven by the fact that >90% of human genes encode transcripts that undergo at least one alternative splicing event with a frequency higher that 10% (1,2). Moreover, alternative splicing contributes to the appropriate spatio-temporal regulation of cellular and developmental processes and to the response to a wide range of extracellular stimuli (3).

A detailed molecular analysis has revealed that alternative splicing decisions involve regulatory sequences, located both in exons and in the flanking introns, which promote (enhancers) or inhibit (silencers) the recognition of splice sites. These elements comprise the target sequences for RNA-binding proteins most of which belong to two groups of widely expressed antagonistic splicing regulatory factors: the SR (serine–arginine-rich) factors that usually promote exon recognition and the group of hnRNP (heterogeneous nuclear ribonucleoprotein) proteins, which in general play an inhibitory role (4). Notably, during tumor progression, stimuli from the tumor microenvironment may affect the expression and/or activity of splicing regulatory factors thus perturbing the physiological splicing program of genes involved in all major aspects of cancer cell biology, including cell cycle control, proliferation, differentiation, signal transduction pathways, cell death, angiogenesis, invasion, motility and metastasis (4–6). In many cases, unscheduled activation of splicing programs typical of embryonic development may occur. However, completely uncharacterized new splicing isoforms are frequently generated as well (7). An increasing body of evidence indicates that splicing variants of many cancer-related genes can directly contribute to the oncogenic phenotype and to the acquisition of resistance to therapeutic treatments (4–6). Hence, understanding the functional role(s) of cancer-associated alternative splicing variants and the mechanisms underlying their production offers the potential to develop novel diagnostic, prognostic and more specific anticancer therapies.

We have contributed to this topic by unveiling the connection between the expression level of splicing factor SRSF1 and the behavior of tumor cells (8). We have shown that SRSF1 (a member of the SR family also known as SF2/ASF) can regulate the epithelial-to-mesenchymal transition (EMT) and the migratory properties of cancer cells (8). EMT is a complex gene expression program through which terminally differentiated epithelial cells acquire mesenchymal features including the ability to efficiently move as single cells through the extracellular matrix (9,10). The EMT program is physiologically important during embryogenesis when it is crucial for organogenesis. However, in adults EMT occurs only during wound healing or it is involved in the metastatic spreading of epithelial cancers (9,10). SRSF1 is an oncoprotein upregulated in many human tumors (11). The involvement of SRSF1 in the EMT program derives from its ability to affect the splicing program of the tyrosine kinase receptor and proto-oncogene Ron. We have shown that SRSF1 promotes the production of ΔRon, a constitutively active isoform, through skipping of exon 11. More specifically, SRSF1 acts by directly binding to an exonic splicing enhancer (ESE) located in the constitutive exon 12 (8). As expected, additional factors such as hnRNP H and A2/B1 have a role in controlling splicing of Ron exon 11 and production of ΔRon. Recently, hnRNP H has been shown to promote the expression of ΔRon by binding to an exonic splicing silencer (ESS) in exon 11, while the mechanism of action of hnRNP A2/B1 has not yet been characterized (12,13). We have previously shown that the activity of the ESE bound by SRSF1 is counteracted by an ESS located upstream of ESE in the same Ron exon 12 (8). However, the molecular mechanism underlying the ability of the ESS to prevent skipping of exon 11 is still unknown.

In this manuscript, we report the characterization of the ESS element in exon 12 of the Ron gene. We show that the ESS is bound by hnRNP A1, a known antagonist of SRSF1 activity in splicing decisions (14). Intriguingly, binding of hnRNP A1 to the ESS sequence in vitro prevents the interaction of SRSF1 to the downstream ESE, which can be relevant for its ability to promote Ron exon 11 inclusion. In addition, hnRNP A1 impacts on the Ron splicing program by regulating the expression level of hnRNP A2/B1, which similarly to SRSF1 promotes ΔRon production (13). Indeed, upregulation of hnRNP A1 induces alternative splicing in the 3′UTR of hnRNP A2/B1 transcripts leading to mRNA degradation via the nonsense-mediated mRNA decay (AS-NMD) pathway (15). Consistently with its ability to inhibit the production of ΔRon, hnRNP A1 activates the reversal of the EMT program, namely the mesenchymal-to-epithelial transition (MET). This activity may be crucial for the malignant process and for the formation of metastases because redifferentiation of mesenchymal cells to an epithelial state is required for the colonization of distant organs (9,10). Altogether, our analysis supports the hypothesis that antagonistic RNA-binding proteins may play an important role in the formation of metastases by modulating a specific splicing event tightly linked to the choice between EMT and MET programs. This will provide new insights toward the development of novel approaches to anticancer therapies.


A comparative morphometric analysis of biodegradable scaffolds as carriers for dental pulp and periosteal stem cells in a model of bone regeneration.
Annibali S, Cicconetti A, Cristalli MP, Giordano G, Trisi P, Pilloni A, Ottolenghi L.
2013 May 24
[Link: PubMed]

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Abstract:
Bone regeneration and bone fixation strategies in dentistry utilize scaffolds containing regenerating-competent cells as a replacement of the missing bone portions and gradually replaced by autologous tissues. Mesenchymal stem cells represent an ideal cell population for scaffold-based tissue engineering. Among them, dental pulp stem cells (DPSCs) and periosteal stem cells (PeSCs) have the potential to differentiate into a variety of cell types including osteocytes, suggesting that they can be used with this purpose. However, data on bone regeneration properties of these types of cells in scaffold-based tissue engineering are yet insufficient.In this study, we evaluated temporal dynamic bone regeneration (measured as a percentage of bone volume on the total area of the defect) induced by DPSCs or PeSCs when seeded with different scaffolds to fill critical calvarial defects in SCID Beige nude mice. Two commercially available scaffolds (granular deproteinized bovine bone with 10% porcine collagen and granular β;-tricalcium phosphate) and one not yet introduced on the market (a sponge of agarose and nanohydroxyapatite) were used. The results showed that tissue-engineered constructs did not significantly improve bone-induced regeneration process when compared with the effect of scaffolds alone. In addition, the data also showed that the regeneration induced by β;-tricalcium phosphate alone was higher after 8 weeks than that of scaffold seeded with the 2 stem cell lines. Altogether these findings suggest that further studies are needed to evaluate the potential of DPSCs and PeSCs in tissue construct and identify the appropriate conditions to generate bone tissue in critical-size defects.


Small molecules affect human dental pulp stem cell properties via multiple signaling pathways.
Al-Habib M, Yu Z, Huang GT.
2013 May 24
[Link: PubMed]

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Abstract:
One fundamental issue regarding stem cells for regenerative medicine is the maintenance of stem cell stemness. The purpose of the study was to test whether small molecules can enhance stem cell properties of mesenchymal stem cells (MSCs) derived from human dental pulp (hDPSCs), which have potential for multiple clinical applications. We identified the effects of small molecules (Pluripotin (SC1), 6-bromoindirubin-3-oxime and rapamycin) on the maintenance of hDPSC properties in vitro and the mechanisms involved in exerting the effects. Primary cultures of hDPSCs were exposed to optimal concentrations of these small molecules. Treated hDPSCs were analyzed for their proliferation, the expression levels of pluripotent and MSC markers, differentiation capacities, and intracellular signaling activations. We found that small molecule treatments decreased cell proliferation and increased the expression of STRO-1, NANOG, OCT4, and SOX2, while diminishing cell differentiation into odonto/osteogenic, adipogenic, and neurogenic lineages in vitro. These effects involved Ras-GAP-, ERK1/2-, and mTOR-signaling pathways, which may preserve the cell self-renewal capacity, while suppressing differentiation. We conclude that small molecules appear to enhance the immature state of hDPSCs in culture, which may be used as a strategy for adult stem cell maintenance and extend their capacity for regenerative applications.


Engineering bone tissue using human dental pulp stem cells and an osteogenic collagen-hydroxyapatite-poly(-lactide-co-{varepsilon}-caprolactone) scaffold.
Akkouch, Adil and Zhang, Ze and Rouabhia, Mahmoud
2013 May 4
[Link: PubMed]

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Abstract:
The aim of this study was to design a new natural/synthetic bioactive bone scaffold for potential use in bone replacement applications. We developed a tri-component osteogenic composite scaffold made of collagen (Coll), hydroxyapatite (HA) and poly(l-lactide-co-ε-caprolactone) (PLCL). This Coll/HA/PLCL composite scaffold was combined with human osteoblast-like cells obtained by differentiation of dental pulp stem cells (DPSCs) to engineer bone tissue in vitro.

Results show that the 3D Coll/HA/PLCL composite scaffold was highly porous, thereby enabling osteoblast-like cell adhesion and growth. Cultured in the Coll/HA/PLCL scaffold, the osteoblast-like cells expressed different osteogenic genes, produced alkaline phosphatase and formed nodules more than did PLCL alone. Micro-CT analyses revealed a significant (30%) increase of tissue mineralisation on the surface as well as inside of the Coll/HA/PLCL scaffold, thus confirming its effectiveness as a bone regeneration platform.


In vitro differentiation into insulin-producing β-cells of stem cells isolated from human amniotic fluid and dental pulp.
Carnevale G, Riccio M, Pisciotta A, Beretti F, Maraldi T, Zavatti M, Cavallini GM, La Sala GB, Ferrari A, De Pol A.
2013 May 3
[Link: PubMed]

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Abstract:
Abstract AIM: To investigate the ability of human amniotic fluid stem cells and human dental pulp stem cells to differentiate into insulin-producing cells. METHODS: Human amniotic fluid stem cells and human dental pulp stem cells were induced to differentiate into pancreatic β-cells by a multistep protocol. Islet-like structures were assessed in differentiated human amniotic fluid stem cells and human dental pulp stem cells after 21 days of culture by dithizone staining. Pancreatic and duodenal homebox-1, insulin and Glut-2 expression were detected by immunofluorescence and confocal microscopy. Insulin secreted from differentiated cells was tested with SELDI-TOF MS and by enzyme-linked immunosorbent assay. RESULTS: Human amniotic fluid stem cells and human dental pulp stem cells, after 7 days of differentiation started to form islet-like structures that became evident after 14 days of induction. SELDI-TOF MS analysis, revealed the presence of insulin in the media of differentiated cells at day 14, further confirmed by enzyme-linked immunosorbent assay after 7, 14 and 21 days. Both stem cell types expressed, after differentiation, pancreatic and duodenal homebox-1, insulin and Glut-2 and were positively stained by dithizone. Either the cytosol to nucleus translocation of pancreatic and duodenal homebox-1, either the expression of insulin, are regulated by glucose concentration changes. Day 21 islet-like structures derived from both human amniotic fluid stem cells and human dental pulp stem cell release insulin in a glucose-dependent manner. CONCLUSION: The present study demonstrates the ability of human amniotic fluid stem cells and human dental pulp stem cell to differentiate into insulin-producing cells, offering a non-pancreatic, low-invasive source of cells for islet regeneration. Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.




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