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[Abstract] Background: Mesenchymal stromal cells (MSCs) have the capacity for self-renewal and multi-differentiation, and for this reason they are considered a potential cellular source in regenerative medicine of cartilage and bone. However, research on this field is impaired by the predisposition of primary MSCs to senescence during culture expansion. Therefore, the aim of this study was to generate and characterize immortalized MSC (iMSC) lines from aged donors. Methods: Primary MSCs were immortalized by transduction of simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). Proliferation, senescence, phenotype and multi-differentiation potential of the resulting iMSC lines were analyzed. Results: MSCs proliferate faster than primary MSCs, overcome senescence and are phenotypically similar to primary MSCs. Nevertheless, their multi-differentiation potential is unbalanced towards the osteogenic lineage. There are no clear differences between osteoarthritis (OA) and non-OA iMSCs in terms of proliferation, senescence, phenotype or differentiation potential. Conclusions: Primary MSCs obtained from elderly patients can be immortalized by transduction of SV40LT and hTERT. The high osteogenic potential of iMSCs converts them into an excellent cellular source to take part in in vitro models to study bone tissue engineering. This research was carried out thanks to the funding from Rede Galega de Terapia Celular 2016 (R2016/036) and Grupos con Potencial de Crecemento 2020 (ED431B 2020/55) from Xunta de Galicia, Proyectos de Investigación 2017 (PI17/02197) from Instituto de Salud Carlos III and the Biomedical Research Network Center (CIBER). The Biomedical Research Network Center (CIBER) is an initiative from Instituto de Salud Carlos III (ISCIII). MPR and SRF were granted a predoctoral fellowship from Xunta de Galicia and European Union (European Social Fund) Xunta de Galicia; R2016/036 Xunta de Galicia; ED431B 2020/55

Reprogramming somatic cells toward pluripotency became possible over a decade ago. Since then, induced pluripotent stem cells (iPSCs) have served as a versatile and powerful tool not only for basic research but also with the long-term goal of using them in human cell transplantation after differentiation. Nonetheless, downstream applications are frequently blurred by the difficulties that researchers have to face when working with iPSCs, such as trouble with clonal selection, in vitro culture and cryopreservation, adaptation to feeder-free conditions, or expansion of the cells. Therefore, in this article we aim to provide other researchers with practical and detailed information to successfully culture and adapt iPSCs. Specifically, we (1) describe the most common problems when in-vitro culturing iPSCs onto feeder cells as well as its possible troubleshooting, and (2) compare different matrices and culture media for adapting the iPSCs to feeder-free conditions. We believe that the troubleshooting and recommendations provided in this article can be of use to other researchers working with iPSCs and who may be experiencing similar issues, hopefully enhancing the appeal of this promising cell source to be used for biomedical investigations, such as tissue engineering or regenerative medicine applications., Graphical abstract, This article contains a descriptive methodological guide on how to culture iPSCs under feeder or feeder-free conditions, paying special attention to provide readers with detailed protocols, tips and tricks, as well as troubleshooting guides than can be of use to other researchers working with iPSCs.

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG [Abstract] Current treatment for corneal endothelial dysfunction consists in the replacement of corneal endothelium by keratoplasty. Owing to the scarcity of donor corneas and the increasing number of transplants, alternative treatments such as cell-based therapies are necessary. In this article, we highlight the biological aspects of the cornea and the corneal endothelium, as well as the context that surrounds the need for new alternatives to conventional keratoplasty. We then review some of those experimental treatments in more detail, focusing on the development of the in vitro and preclinical phases of two cell-based therapies: tissue-engineered endothelial keratoplasty (TE-EK) and cell injection. In the case of TE-EK graft construction, we analyse the current progress, considering all the requirements it must meet in order to be functional. Moreover, we discuss the inherent drawbacks of endothelial keratoplasties, which TE-EK grafts should overcome in order to make surgical intervention easier and to improve the outcomes of current endothelial keratoplasties. Finally, we analyse the development of preclinical trials and their limitations in terms of performing an optimal functional evaluation of cell-based therapy, and we conclude by discussing early clinical trials in humans. Xunta de Galicia; R2016/036 Xunta de Galicia; ED431B 2020/55 Xunta de Galicia; ED481B 2017/029 Xunta de Galicia; ED481A-2019/206 Xunta de Galicia; ED481A-2017/280 This work was carried out thanks to funding from the Rede Galega de Terapia Celular 2016 (R2016/036) and Grupos con Potencial de Crecemento 2020 (ED431B 2020/55) both from Xunta de Galicia. This work was supported by one postdoctoral and two predoctoral fellowships from the Xunta de Galicia and the European Union (European Regional Development Fund) [grant numbers ED481B 2017/029, ED481A-2019/206, and ED481A-2017/280, respectively], as well as by two predoctoral fellowships for research stays from INDITEX-University of A Coruña-2019.

[Abstract] Induced pluripotent stem cells (iPSCs) represent an unlimited source of pluripotent cells capable of di erentiating into any cell type of the body. Several studies have demonstrated the valuable use of iPSCs as a tool for studying the molecular and cellular mechanisms underlying disorders a ecting bone, cartilage and muscle, as well as their potential for tissue repair. Musculoskeletal diseases are one of the major causes of disability worldwide and impose an important socio-economic burden. To date there is neither cure nor proven approach for e ectively treating most of these conditions and therefore new strategies involving the use of cells have been increasingly investigated in the recent years. Nevertheless, some limitations related to the safety and di erentiation protocols among others remain, which humpers the translational application of these strategies. Nonetheless, the potential is indisputable and iPSCs are likely to be a source of di erent types of cells useful in the musculoskeletal field, for either disease modeling or regenerative medicine. In this review, we aim to illustrate the great potential of iPSCs by summarizing and discussing the in vitro tissue regeneration preclinical studies that have been carried out in the musculoskeletal field by using iPSCs. Instituto de Salud Carlos III; PI17/02197 Xunta de Galicia; R2016/036 Xunta de Galicia; R2014/050 Xunta de Galicia; CN2012/142 Xunta de Galicia; GPC2014/048

[Abstract] Human bone marrow-derived mesenchymal stromal cells (MSCs) obtained from aged patients are prone to senesce and diminish their differentiation potential, therefore limiting their usefulness for osteochondral regenerative medicine approaches or to study age-related diseases, such as osteoarthiritis (OA). MSCs can be transduced with immortalizing genes to overcome this limitation, but transduction of primary slow-dividing cells has proven to be challenging. Methods for enhancing transduction efficiency (such as spinoculation, chemical adjuvants, or transgene expression inductors) can be used, but several parameters must be adapted for each transduction system. In order to develop a transduction method suitable for the immortalization of MSCs from aged donors, we used a spinoculation method. Incubation parameters of packaging cells, speed and time of centrifugation, and valproic acid concentration to induce transgene expression have been adjusted. In this way, four immortalized MSC lines (iMSC#6, iMSC#8, iMSC#9, and iMSC#10) were generated. These immortalized MSCs (iMSCs) were capable of bypassing senescence and proliferating at a higher rate than primary MSCs. Characterization of iMSCs showed that these cells kept the expression of mesenchymal surface markers and were able to differentiate towards osteoblasts, adipocytes, and chondrocytes. Nevertheless, alterations in the CD105 expression and a switch of cell fate-commitment towards the osteogenic lineage have been noticed. In conclusion, the developed transduction method is suitable for the immortalization of MSCs derived from aged donors. The generated iMSC lines maintain essential mesenchymal features and are expected to be useful tools for the bone and cartilage regenerative medicine research. Xunta de Galicia; R2016/036 Xunta de Galicia; R2014/050 Xunta de Galicia; CN2012/142 Xunta de Galicia; GPC2014/048 Deputación da Coruña; BINV-CS/2016 Instituto de Salud Carlos III; PI17/02197

Here, we report the establishment of the human iPS cell line N1-FiPS4F#7 generated from skin cells of a patient with no rheumatic diseases, thus obtaining an appropriate control iPS cell line for researchers working in the field of rheumatic diseases. The reprogramming factors Oct4, Sox2, Klf4 and c-Myc were introduced using a non-integrating reprogramming strategy involving Sendai Virus. Keywords: Human iPS, Reprogramming, Sendai virus, Pluripotency, Cell line, Embryoid body

[Abstract] The establishment of cartilage regenerative medicine is an important clinical issue, but the search for cell sources able to restore cartilage integrity proves to be challenging. Human mesenchymal stromal cells (MSCs) are prone to form epiphyseal or hypertrophic cartilage and have an age-related limited proliferation. On the other hand, it is difficult to obtain functional chondrocytes from human embryonic stem cells (ESCs). Moreover, the ethical issues associated with human ESCs are an additional disadvantage of using such cells. Since their discovery in 2006, induced pluripotent stems cells (iPSCs) have opened many gateways to regenerative medicine research, especially in cartilage tissue engineering therapies. iPSCs have the capacity to overcome limitations associated with current cell sources since large numbers of autologous cells can be derived from small starting populations. Moreover, problems associated with epiphyseal or hypertrophic-cartilage formation can be overcome using iPSCs. iPSCs emerge as a promising cell source for treating cartilage defects and have the potential to be used in the clinical field. For this purpose, robust protocols to induce chondrogenesis, both in vitro an in vivo, are required. This review summarises the recent progress in iPSC technology and its applications for cartilage repair. Xunta de Galicia; R2016/036 Xunta de Galicia; R2014/050 Xunta de Galicia; GPC2014/048 Instituto de Salud Carlos III; PI17/02197

[Abstract] Healthy cartilage maintenance relies on an equilibrium among the anabolic and catabolic processes in chondrocytes. With the onset of osteoarthritis (OA), increased interleukin (IL)-1β levels induce an inhibition of the synthesis of extracellular matrix (ECM) proteins, as well as an increase in proteases. This eventually leads to a predominance of the catabolic phenotype and the progressive loss of articular cartilage. Hydrogen sulfide (H2S) is a small gaseous molecule recognized as the third endogenous gasotransmitter. When administered exogenously, it has shown anti-inflammatory and anti-catabolic properties in several in vitro and in vivo models. Here, OA cartilage disks were co-cultured in vitro with IL-1β (5 ng/ml) and NaSH or GYY4137 (200 or 1000 μM) for 21 days. The ability of these two H2S-producing compounds to avoid long term extracellular matrix (ECM) destruction was evaluated. We used a glycosaminoglycan (GAG) quantification kit histology and immunohistochemistry (IHC) to evaluate matrix proteins degradation and matrix metalloproteinases (MMP) abundance. Through the GAGs quantification assay, safranin O (S-O) and toluidine blue (TB) stains, and keratan/chondroitin sulfate (KS/ChS) IHCs it was shown that co-stimulation with H2S-forming reagents effectively avoided GAGs destruction. Both Masson's trichrome (MT) stain and collagen (col) type II IHC, as well as aggrecan (agg) IHC demonstrated that not only were these proteins protected but even promoted, their abundance being higher than in the basal condition. Further, stains also demonstrated that positivity in the inter-territorial and intra-cellular for the different matrix components were rescued, suggesting that NaSH and GYY4137 might also have pro-anabolic effects. In addition, a clear protective effect against the increased MMPs levels was seen, since increased MMP3 and 13 levels were subsequently reduced with the co-stimulation with sulfide compounds. In general, GYY4137 was more effective than NaSH, and increasing the dose improved the results. This study demonstrates that H2S anti-catabolic effects, which had been previously proven in short-term (24–48 h) in vitro cellular models, are maintained over time directly in OA cartilage tissue. Instituto de Salud Carlos III; PI12/00329

[Abstract] Knowledge and research results about hand osteoarthritis (hOA) are limited due to the lack of samples and animal models of the disease. Here, we report the generation of two induced pluripotent stem cell (iPSC)-lines from patients with radiographic hOA. Furthermore, we wondered whether these iPSC-lines carried single nucleotide polymorphisms (SNPs) within genes that have been associated with hOA. Finally, we performed chondrogenic differentiation of the iPSCs in order to prove their usefulness as cellular models of the disease. We performed a non-integrative reprogramming of dermal fibroblasts obtained from two patients with radiographic rhizarthrosis and non-erosive hOA by introducing the transcriptional factors Oct4, Sox2, Klf4 and c-Myc using Sendai virus. After reprogramming, embryonic stem cell-like colonies emerged in culture, which fulfilled all the criteria to be considered iPSCs. Both iPSC-lines carried variants associated with hOA in the four studied genes and showed differences in their chondrogenic capacity when compared with a healthy control iPSC-line. To our knowledge this is the first time that the generation of iPSC-lines from patients with rhizarthrosis and non-erosive hOA is reported. The obtained iPSC-lines might enable us to model the disease in vitro, and to deeper study both the molecular and cellular mechanisms underlying hOA. This study was carried out thanks to the funding from Fundación Española de Reumatología (Proyectos 2014), Proyectos de Investigación 2016 (PI16/02124) and 2017 (PI17/02197) from Instituto de Salud Carlos III-General Subdirection of Assesment and Promotion of the Research – European Regional Development Fund (FEDER) “A way of making Europe”, Rede Galega de Terapia Celular and Grupos con Potencial de Crecemento, Xunta de Galicia (R2016/036, R2014/050, CN2012/142 and GPC2014/048); Deputación da Coruña (BINV-CS/2015); University of A Coruña; Centro de Investigación Biomédica en Red-Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN). Rocío Castro-Viñuelas, María Piñeiro-Ramil and Silvia Rodríguez-Fernández are granted by a predoctoral fellowship from Xunta de Galicia and European Union (European Social Fund) and Clara Sanjurjo-Rodríguez is beneficiary of a postdoctoral fellowship from Xunta de Galicia Xunta de Galicia; R2016/036 Deputación da Coruña; BINV-CS/2015 Xunta de Galicia; R2014/050 Xunta de Galicia; CN2012/142 Xunta de Galicia; GPC2014/048

[Abstract] This work proposes a method based on image analysis and machine and statistical learning to model and estimate osteocyte growth (in type I collagen scaffolds for bone regeneration systems) and the collagen degradation degree due to cellular growth. To achieve these aims, the mass of collagen -subjected to the action of osteocyte growth and differentiation from stem cells- was measured on 3 days during each of 2 months, under conditions simulating a tissue in the human body. In addition, optical microscopy was applied to obtain information about cellular growth, cellular differentiation, and collagen degradation. Our first contribution consists of the application of a supervised classification random forest algorithm to image texture features (the structure tensor and entropy) for estimating the different regions of interest in an image obtained by optical microscopy: the extracellular matrix, collagen, and image background, and nuclei. Then, extracellular-matrix and collagen regions of interest were determined by the extraction of features related to the progression of the cellular growth and collagen degradation (e.g., mean area of objects and the mode of an intensity histogram). Finally, these critical features were statistically modeled depending on time via nonparametric and parametric linear and nonlinear models such as those based on logistic functions. Namely, the parametric logistic mixture models provided a way to identify and model the degradation due to biological activity by estimating the corresponding proportion of mass loss. The relation between osteocyte growth and differentiation from stem cells, on the one hand, and collagen degradation, on the other hand, was determined too and modeled through analysis of image objects’ circularity and area, in addition to collagen mass loss. This set of imaging techniques, machine learning procedures, and statistical tools allowed us to characterize and parameterize type I collagen biodegradation when collagen acts as a scaffold in bone regeneration tasks. Namely, the parametric logistic mixture models provided a way to identify and model the degradation due to biological activity and thus to estimate the corresponding proportion of mass loss. Moreover, the proposed methodology can help to estimate the degradation degree of scaffolds from the information obtained by optical microscopy. Ministerio de Asuntos Económicos y Transformación Digital; MTM2014-52876-R Ministerio de Asuntos Económicos y Transformación Digital; MTM2017-82724-R Xunta de Galicia; ED431C-2016-015 Xunta de Galicia; ED431G/01

Background: Bone-marrow mesenchymal stromal cells (MSCs) are multipotent self-renewal adult cells with high potential to regenerate the damaged tissues in degenerative diseases such as osteoarthritis (OA). Nevertheless, their usefulness for osteochondral Regenerative Medicine research is hampered by their proneness to senescence when in vitro cultured. Currently, MSC lines available are scarce and present limitations regarding their differentiation capacities. In addition, there is none OA MSC line available for research on this disease. Objectives: The aim of this study was to generate and characterize immortalized human OA and non-OA MSC lines for their use in osteochondral Regenerative Medicine research. Methods: For the generation of the immortalized MSC lines, SV40 large T antigen (SV40LT) and GFP-fused human telomerase reverse transcriptase (hTERT) were used. Primary MSCs derived from two hip OA patients and one hip fracture patient without OA were transduced by spinoculation at 800 xg for 45 minutes. Transgene expression was induced by valproic acid. Nuclear expression of SV40LT and GFP was tested by immunofluorescence. Proliferation and senescence were investigated through calculation of population doublings (PDs) at each passage after immortalization and β-galactosidase staining after 100 PDs for each MSC line. Maintenance of MSC characteristics in immortalized MSCs was tested by analysis of CD29, CD44, CD73, CD90, CD105, CD34 and CD45 expression by flow cytometry and cell differentiation experiments. Multi-lineage differentiation potential was analysed histochemical, immunohistochemical and molecularly. Results: Three MSC lines have been generated: two OA and one non-OA. As shown by immunofluorescence, SV40LT is expressed in the nucleoplasm of these cells, while GFP-fused hTERT is expressed in the nucleoli. A constant proliferation rate thoroughout subculturing in addition to β-galactosidase negative staining confirms that immortalized MSC lines do not senesce, unlike primary MSCs. Expression of CD29, CD44, CD73 and CD90 and lack of CD34 and CD45 was conserved in immortalized MSC lines, while CD105 expression was altered for transduction status and passage. Both OA and non-OA immortalized MSC lines maintain their multipotency (namely, osteogenic, chondrogenic and adipogenic differentiation capacity). Conclusion: Both OA and non-OA MSCs are susceptible to immortalization by SV40LT and hTERT. For they increased lifespan combined with keeping of most of primary MSC characteristics, these MSC lines are expected to be valuable tools for the research on Regenerative Medicine for OA as part of Tissue Engineering in vitro models. Acknowledgement: Xunta de Galicia and European Union (European Social Fund-ESF); Deputacion da Coruna; Rede de Investigacion en Celulas Nai e Terapia Celular (R2016/036, R2014/050 and CN2012/142) and Grupos con Potencial de Crecemento (GPC2014/048) (Xunta de Galicia); CIBER-BNN; Fundacion Espanola de Reumatologia (FER); Fondo de Investigacion en Salud (FIS) (PI17/02197). Disclosure of Interests: Maria Pineiro-Ramil: None declared, Rocio Castro-Vinuelas: None declared, Clara Sanjurjo-Rodriguez: None declared, Silvia Rodriguez-Fernandez: None declared, Tamara Hermida Gomez: None declared, Francisco Javier de-Toro-Santos: None declared, Francisco J. Blanco Consultant for: AbbVie, Bioiberica, BMS, GSK, Grunenthal, Janssen, Lilly, Pfizer, Regeneron, Roche, Sanofi, TRB Chemedica, and UCB, Isaac Fuentes-Boquete: None declared, SILVIA MARIA DiAZ-PRADO: None declared

Background Research results in the field of hand OA are currently limited due to the unavailability of tissue samples and lack of animal models replicating the features of the disease in humans. Cellular in vitro models are important tools to elucidate molecular mechanisms and pathways that are involved in hand OA. Specifically, induced pluripotent stem cells (iPSc) are considered ideal tools for this purpose since they allow the use of unlimited cells with chondrogenic differentiation potential. However, there are not studies published generating iPSc from patients with hand OA. Objectives To generate and characterize iPSc-lines from patients with radiographic hand OA and healthy donors, which can be used as cellular models of the disease, for studying the pathogenesis of the disease in vitro and for testing new drugs. Methods Patients with hand OA (non erosive hand OA with right thumb OA) and a healthy control were selected for the study. Using the explant culture technique, fibroblasts from skin biopsies of these patients were isolated. Transcriptional factors Oct4, Sox2, Klf4 and c-Myc were used for the reprogramming process; which was performed by using a non-integrating method, the Sendai virus. Cell lines obtained were morphologically, phenotypically and functionally characterized. To evaluate weather these iPSc lines could be used as cellular model of hand OA, presence of single nucleotide polymorphisms (SNPs) within the genes ALDH1A2 and SMAD3 were studied by Sanger sequencing, before and after reprogramming. Variants rs3204689 and rs12901499 respectively have been associated with severe OA of the hand (Styrkarsdottir et al., 2014; Shu-Thao Gao et al., 2018). Finally, chondrogenic differentiation capacity of the “healthy” and “ill” iPSc-lines was studied by means of histological techniques. Results Fibroblasts were isolated from one patient with radiographic hand OA and one healthy donor. Three weeks after reprogramming, embryonic stem cell-like colonies emerged in culture. These cells showed positivity for alkaline phosphatase activity (fig. 1A) and the pluripotency markers Tra1-81 and Nanog (fig. 1B). Molecular analyses showed high relative expression levels of the pluripotency-related genes OCT4, SOX2, NANOG and CRIPTO in the iPSc. These cells were also able to give rise to cells from the three germ layers (fig. 1C). Indeed, during mesodermal differentiation, spontaneously beating cardiomyocytes were seen in culture. Regarding SNPs studies, cells from the patient with hand OA were homozygous for the at-risk allele in both genes studied, both before and after reprogramming (fig. 1D). The “ill” iPSc-line (MOAFiPS 15/645#7) showed worse chondrogenic differentiation than the “healthy” iPSc-line (NFiPS 15/637#7), as shown by the micromasses collagen and proteoglycan content (fig. 1E). Conclusion The generation of one iPSc-line form patients with hand OA is reported for the first time. The presence of the at-risk alleles within the ALDH1A2 and SMAD3 genes were maintained after fibroblast reprogramming. The iPSC lines obtained shown differences in their chondrogenic differentiation capacity, showing their usefulness to model hand OA in vitro, and to deeper study the role of these genetic variants in the pathogenesis of hand OA. References [1] Styrkarsdottir U, et al. (2014). Nat Genet. 46(5):498-502. [2] Shu-Tao Gao, et al. (2018) Ther Clin Risk Manag. 14: 929–936. Acknowledgement FER; ISCIII (PI17/02197); CIBER-BBN; REDICENT; GPC (Xunta de Galicia); Diputacion Coruna; Xunta de Galicia y Fondo Social Europeo, Servicio de Genetica Hospital Teresa Herrera; Servicio Radiofisica Centro Oncologico de Galicia, Universidade da Coruna. Disclosure of Interests Rocio Castro-Vinuelas: None declared, Clara Sanjurjo-Rodriguez: None declared, Maria Pineiro-Ramil: None declared, Tamara Hermida Gomez: None declared, Isaac Fuentes-Boquete: None declared, Javier de Toro-Santos: None declared, Francisco J. Blanco Consultant for: AbbVie, Bioiberica, BMS, GSK, Grunenthal, Janssen, Lilly, Pfizer, Regeneron, Roche, Sanofi, TRB Chemedica, and UCB, SILVIA MARIA DIAZ-PRADO: None declared

[Abstract] This methodology permits to simulate the performance of different Poly(D,L-lactide-co-glycolide) copolymer formulations (PDLGA) in the human body, to identify the more influencing variables on hydrolytic degradation and, thus, to estimate biopolymer degradation level. The PDLGA characteristic degradation trends, caused by hydrolysis processes, have been studied to define their future biomedical applications as dental scaffolds. For this purpose, the mass loss, pH, glass transition temperature (Tg) and absorbed water mass of the different biopolymers have been obtained from samples into a phosphate-buffered saline solution (PBS) with initial pH of 7.4, at 37°C (human body conditions). The mass loss has been defined as the variable that characterize the biopolymer degradation level. Its dependence relationship with respect to time, pH and biopolymer formulation has been modelled using statistical learning tools. Namely, generalized additive models (GAM) and nonlinear mixed-effects regression with logistic and asymptotic functions have been applied. GAM model provides information about the relevant variables and the parametric functions that relate mass loss, pH and time. Mixed effects are introduced to model and estimate the degradation properties, and to compare the PDLGA biopolymer populations. The degradation path for each polymer formulation has been estimated and compared with respect to the others for helping to use the proper polymer for each specific medical application, performing selection criteria. It was found that the mass loss differences in PDLGA copolymers are strongly related with the way the pH decay versus time, due to carboxylic acid groups formation. This may occur in those environments in which the degradation products remain relatively confined with the non degraded mass. This is the case emulated with the present experimental procedure. The results show that PDLGA polymers degradation degree, in terms of half life and degradation rate, is increasing when acid termination is included, when DL-lactide molar ratio is reduced, decreasing the midpoint viscosity, or when glycolide is not included. Xunta de Galicia; ED431G/012016-2019 Xunta de Galicia; ED431C2016-015 Ministerio de Economía y Competitividad; MTM2014-52876-R

Background To date, there is no drug able cure such a prevalent disorder as it is hand osteoarthritis (OA). Cell therapies using stem cells have emerged as a promising strategy to explore and develop new treatments. Specifically, induced pluripotent stem cells (iPSc) are considered ideal tools not only for this purpose, but also for modelling the disease. The advantages of using an established iPSc line are unlimited cell source with regeneration capacity and chondrogenic differentiation potential. However, there are not many studies published generating iPSc from patients with hand OA. Objectives The aim of this study has been to generate an iPSc-line from human fibroblasts obtained from patients with radiographic hand OA, which can be useful for drug discovery, disease modelling and regenerative medicine applications. Methods Patients with radiographic hand OA and a healthy control were selected for the study. Using the explant culture technique, fibroblasts from 3 mm skin biopsies of these patients were isolated. These cells were histologically characterised and positivity for fibroblast markers was quantified. These cells were also karyotyped in order to confirm that chromosomal abnormalities did not exist before reprogramming. Four transcriptional factors were used for the reprogramming process: Oct4, Sox2, Klf4 and c-Myc. These were delivered using a non-integrative methodology that involves the use of Sendai virus. Cell lines obtained were clonally expanded over 20 passages and characterised for pluripotency markers by immunohistochemistry and RT-PCR, before and after reprogramming (figure 1). The best two clones of each one of the patients were selected according to the expression of the pluripotency markers to establish the cell-line. Results Cells were isolated from skin biopsies of two patients with radiographic hand OA and one healthy donor. Histological and immunohistochemical analyses showed that the 85%–95% of cells in the culture were fibroblasts, which presented a normal karyotype: 46, XX. Three weeks after reprogramming, embryonic stem cell-like colonies emerged in culture. These cells were positivity stained for alkaline phosphatase activity and the pluripotency markers Tra1–81 and Nanog. Alkaline phosphatase staining shows in blue the colonies correctly reprogrammed. Molecular analyses showed high relative expression levels of the endogenous pluripotency associated genes OCT4, SOX2, KLF4, NANOG and CRIPTO in the iPSc, but not in the no-reprogrammed fibroblasts. No presence of Sendai Virus-related genes was detected. Conclusions Fibroblasts were successfully isolated from all the patients. The reprogramming process using Sendai virus enabled us to generate iPSc from two patients with radiographic hand OA and one healthy donor. Acknowledgements Fundacion Espanola de Reumatologia; CIBER-BBN; REDICENT; GPC (Xunta de Galicia); Diputacion da Coruna; Xunta de Galicia y Fondo Social Europeo, Servicio de genetica del Hospital Teresa Herrera; Servicio de Radiofisica del Centro Oncologico de Galicia, Universidade da Coruna. Disclosure of Interest None declared

[Abstract] The integrity of the articular cartilage is necessary for the proper functioning of the diarthrodial joint. The self-repair capacity of this tissue is very limited and, currently, there is no effective treatment capable of restoring it. The degradation of the articular cartilage leads to osteoarthritis (OA), a leading cause of pain and disability mainly among older people. Different cell treatments have been developed with the aim of forming a repair tissue with the characteristics of native articular cartilage, including cellular therapy and tissue engineering. Cell therapy-based approaches include bone marrow-stimulating techniques, implants of periosteum and perichondrium, ostechondral grafting and implantation of chondrogenic cells as chondrocytes, mesenchymal stem cells or induced pluripotent stem cells. In tissue engineering-based approaches cell-free scaffolds capable of recruiting endogenous cells or chondrogenic cell-loaded scaffolds may be used. However, despite the numerous treatments available nowadays, no technique has been able to consistently regenerate native articular cartilage in clinical trials. Although many cell therapy and tissue engineering studies have shown promising results and clinical improvement, these treatments generate a fibrocartilaginous tissue different from native articular cartilage. More research is needed to improve cell-based approaches and prove its efficacy. Xunta de Galicia; R2014/050 Ministerio de Economía y Competitividad; RTC-2016-5386-1

[Abstract] The poly(DL-lactide-co-glycolide) (PDLGA) copolymers have been specifically designed and performed as biomaterials, taking into account their biodegradability and biocompatibility properties. One of the applications of statistical degradation models in material engineering is the estimation of the materials degradation level and reliability. In some reliability studies, as the present case, it is possible to measure physical degradation (mass loss, water absorbance, pH) depending on time. To this aim, we propose an expert system able to provide support in collagen degradation analysis through computer vision methods and statistical modelling techniques. On this base, the researchers can determine which statistical model describes in a better way the biomaterial behaviour. The expert system was trained and evaluated with a corpus of 63 images (2D photographs obtained by electron microscopy) of human mesenchymal stem cells (CMMh-3A6) cultivated in a laboratory experiment lasting 44 days. The collagen type-1 sponges were arranged in 3 groups of 21 samples (each image was obtained in intervals of 72 hours).

[Abstract] The Poly(DL-lactide-co-glycolide) copolymers (PDLGA) have designed and performed as biomaterials, taking into account their biodegradability and biocompatibility properties. These materials have a wide range of application in medicine such as orthopedic implants, general surgical implants (suture materials), osteosynthesis, bone cement, among many others. For these reasons, in this paper, we present an intelligent educational environment that can be used for both, researchers and students interested in the analysis of the biomaterial behavior under certain conditions. Our platform includes a Learning Objects (LOs) for MOODLE, and in the same way, implements several digital image processing techniques as well as a decision support module based on a random forest algorithm and statistical modeling. With the aim of determining the real feasibility of this proposal, we have presented the system to 34 Ecuadorian engineering students. After testing the platform, the students answered a survey aimed at determining their perception of the system. The results provide several guidelines to continue with the developing of the platform.

[Abstract] The purpose of this study was to investigate cartilage repair of in vitro lesion models using human bone marrow mesenchymal stromal cells (hBMSCs) with different collagen (Col) scaffolds. Lesions were made in human cartilage biopsies. Injured samples were pre-treated with interleukin 1β (IL1β) for 24 h; also, samples were not pre-treated. hBMSCs were seeded on different types of collagen scaffolds. The resulting constructs were placed into the lesions, and the biopsies were cultured for 2 months in chondrogenic medium. Using the modified ICRSII scale, neotissues from the different scaffolds showed ICRS II overall assessment scores ranging from 50% (fibrocartilage) to 100% (hyaline cartilage), except for the Col I +Col II +HS constructs (fibrocartilage/hyaline cartilage, 73%). Data showed that hBMSCs cultured only on Col I +Col II +HS scaffolds displayed a chondrocyte-like morphology and cartilage-like matrix close to native cartilage. Furthermore, IL1β pre-treated biopsies decreased capacity for repair by hBMSCs and decreased levels of chondrogenic phenotype of human cartilage lesions. Instituto de Salud Carlos III; CB06/01/0040 Xunta de Galicia ; R2016/036 Xunta de Galicia; R2014/050 Xunta de Galicia; GPC2014/048 Ministerio de Esconomía, Industria y Competitividad; RTC-2016-5386-1 Madrid (Comunidad Autónoma); S2009/MAT-1472

[Abstract] The unavailability of sufficient numbers of human primary cells is a major roadblock for in vitro repair of bone and/or cartilage, and for performing disease modelling experiments. Immortalized mesenchymal stromal cells (iMSCs) may be employed as a research tool for avoiding these problems. The purpose of this review was to revise the available literature on the characteristics of the iMSC lines, paying special attention to the maintenance of the phenotype of the primary cells from which they were derived, and whether they are effectively useful for in vitro disease modeling and cell therapy purposes. This review was performed by searching on Web of Science, Scopus, and PubMed databases from 1 January 2015 to 30 September 2019. The keywords used were ALL = (mesenchymal AND (“cell line” OR immortal*) AND (cartilage OR chondrogenesis OR bone OR osteogenesis) AND human). Only original research studies in which a human iMSC line was employed for osteogenesis or chondrogenesis experiments were included. After describing the success of the immortalization protocol, we focused on the iMSCs maintenance of the parental phenotype and multipotency. According to the literature revised, it seems that the maintenance of these characteristics is not guaranteed by immortalization, and that careful selection and validation of clones with particular characteristics is necessary for taking advantage of the full potential of iMSC to be employed in bone and cartilage-related research. Xunta de Galicia; R2016/036 Deputación da Coruña; BINV-CS/2016 Xunta de Galicia; R2014/050 Xunta de Galicia; CN2012/142 Xunta de Galicia; GPC2014/048

The aim of this study was to evaluate proliferation and chondrogenic differentiation of human bone-marrow mesenchymal stromal cells (hBMSCs) cultured on collagen biomaterials.hBMSCs were seeded on five different collagen (Col) sponges: C1C2 (types I and II Col), C1C2HS (types I and II Col plus heparan sulphate (HS)), C1C2CHS (types I and II Col plus chondroitin sulphate (CHS)), C1-OLH3 (type I Col plus low molecular weight heparin) and C1CHS (type I Col plus CHS). The resulting constructs were analyzed by histological and immunohistochemical staining, molecular biology and electron microscopy. Col released into culture media was measured by a dye-binding method Results: hBMSCs on biomaterials C1C2, C1C2HS and C1C2CHS had more capacity to attach, proliferate and synthesize Col II and proteoglycans in the extracellular matrix (ECM) than on C1-OLH3 and C1CHS. The presence of aggrecan was detected only at the gene level. Total Col liberated by the cells in the supernatants in all scaffold cultures was detected. The level of Col I in the ECM was lower in C1-OLH3 and that of Col II was highest in C1C2 and C1C2HS. Electron microscopy showed differently shaped cells, from rounded to flattened, in all constructs. Col fibers in bundles were observed in C1C2CHS by transmission electron microscopy.The results show that Col I and Col II (C1C2, C1C2HS and C1C2CHS) biomaterials allowed cell proliferation and chondrogenic-like differentiation of hBMSCs at an early stage. Constructs cultured on C1C2HS and C1C2CHS showed better cartilage-like phenotype than the other ones.

[Abstract] Objective. Identify candidate SEBs (surrogate endpoint biomarkers) for premalignant trends in head and neck mucosa. Study design. Study, by qPCR (quantitative real-time polymerase chain reaction), the expression of COX-2, EGFR and p53 in 24 biopsies of non-dysplastic oral leukoplakia and contra-lateral normal-appearing mucosa. Results. COX-2 was up-regulated in leukoplakia (79.2%); whereas EGFR and p53 were up-regulated (p > 0.05) in oral contra-lateral normal-appearing mucosa (60% and 46% respectively). Also, p53 expression was correlated with tobacco smoke habits and Spearman's rank correlation coefficient showed a positive linear correlation between p53 and EGFR mRNA expression levels. Conclusions. COX-2 would serve as SEB of oral leukoplakia. The results suggest that p53 appears to be one of the molecular targets of tobacco-related carcinogens in leukoplakia and that the co-expression of p53 and EGFR may play a role in this kind of oral pre-cancerous lesion. More detailed studies of EGFR and p53 should be continued in the future.

[Abstract] Background. The use of autologous or allogenic stem cells has recently been suggested as an alternative therapeutic approach for treatment of cartilage defects. Bone marrow mesenchymal stem cells (BM-MSCs) are well-characterized multipotent cells that can differentiate into different cell types. Understanding the potential of these cells and the molecular mechanisms underlying their differentiation should lead to innovative protocols for clinical applications. The aim of this study was to evaluate the usefulness of surface antigen selection of BM-MSCs and to understand the mechanisms underlying their differentiation. Methods. MSCs were isolated from BM stroma and expanded. CD105+ subpopulation was isolated using a magnetic separator. We compared culture-expanded selected cells with non-selected cells. We analyzed the phenotypic profiles, the expression of the stem cell marker genes Nanog, Oct3/4, and Sox2 and the multi-lineage differentiation potential (adipogenic, osteogenic, and chondrogenic). The multi-lineage differentiation was confirmed using histochemistry, immunohistochemistry and/or real-time polymerase chain reaction (qPCR) techniques. Results. The selected and non-selected cells displayed similar phenotypes and multi-lineage differentiation potentials. Analyzing each cell source individually, we could divide the six donors into two groups: one with a high percentage of CD29 (β1-integrin) expression (HL); one with a low percentage of CD29 (LL). These two groups had different chondrogenic capacities and different expression levels of the stem cell marker genes. Conclusions. This study showed that phenotypic profiles of donors were related to the chondrogenic potential of human BM-MSCs. The chondrogenic potential of donors was related to CD29 expression levels. The high expression of CD29 antigen seemed necessary for chondrogenic differentiation. Further investigation into the mechanisms responsible for these differences in BM-MSCs chondrogenesis is therefore warranted. Understanding the mechanisms for these differences will contribute to improved clinical use of autologous human BM-MSCs for articular cartilage repair. Servizo Galego de Saúde; PS07/84 Instituto de Salud Carlos III; CIBER BBN CB06-01-0040

[Abstract] Genes of the Wnt and Frizzled class, expressed in HNSCC tissue and cell lines, have an established role in cell morphogenesis and differentiation, and also they have oncogenic properties. We studied Wnt and Fz genes as potential tumor-associated markers in HNSCC by qPCR. Expression levels of Wnt and Fz genes in 22 unique frozen samples from HNSCC were measured. We also assessed possible correlation between the expression levels obtained in cancer samples in relation to clinicopathologic outcome. Wnt-1 was not expressed in the majority of the HNSCC studied, whereas Wnt-5A was the most strongly expressed by the malignant tumors. Wnt-10B expression levels were related with higher grade of undifferentiation. Related to Fz genes, Fz-5 showed more expression levels in no-affectation of regional lymph nodes. Kaplan–Meier survival analyses suggest a reduced time of survival for low and high expression of Wnt-7A and Fz-5 mRNA, respectively. qPCR demonstrated that HNSCC express Wnt and Fz members, and suggested that Wnt and Fz signaling is activated in HNSCC cells.

Articular cartilage lesions which do not affect the integrity of subchondral bone, they are not able to repair it expontaneously. The asymptomatic nature of these lesions induces articular cartilage degeneration and development of an arthrosic process. To avoid the necessity to receive joint replacement surgery, it has been developed different treatments of cellular therapy which are focused to create new tissues whose structure, biochemistry composition and function will be the same than native articular cartilage. Approaches used to access the stream produce a fibrocartilaginose tissue which is not an articular cartilage. Implantation of autologous chondrocytes and autologous mosaicplasties induces a quality better articular cartilage. Furthermore both techniques involve damage in the sane cartilage; because of trying to get a big amount of chondrocytes or because of extraction osteochondral cylinder which will be implanted in the injured joint. The stem cells are a promising toll to repair articular cartilage, however they are in a previous experimentation step yet. Although the present studies using cellular therapy improves clinically and functionally, it is not able to regenerate an articular cartilage which offer resistance the degeneration process.

[Abstract] Mesenchymal stem cells (MSCs) are an accepted candidate for cell-based therapy of multiple diseases. The interest in MSCs and their possible application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently, like aggregation and transforming growth factor beta (TGFβ) delivery, hypoxia has been indicated as crucial for complete chondrogenesis. The aim of this study was to test different culture conditions for directing stem cell differentiation into the chondrogenic lineage in vitro by testing different TGFβ superfamily members into the culture media under normoxic conditions. All chondrogenic culture conditions used allowed the differentiation of bone marrow-MSCs (BM-MSCs) into chondrogenic lineage. Chondrogenic induction capacity depended on the growth factor added to the culture media. In particular, the chondrogenic culture condition that better induced chondrogenesis was the medium that included the combination of three growth factors: bone morphogenetic protein-2 (BMP-2), BMP-7 and TGFβ-3. In this culture media, differentiated cells showed the highest levels expression of two markers of chondrogenesis, SOX9 and COL2A1, compared to the control points (p < 0.05, two-tailed t test). In our experimental conditions, the combination of BMP-2, BMP-7 and TGFβ-3 was the most effective in promoting chondrogenesis of BM-MSCs. These results underline the importance of determining in each experimental design the best protocol for in vitro directing stem cell differentiation into the chondrogenic lineage. Galicia. Consellería de Sanidade; PS07/84 Ministerio de Ciencia e Innovacion; PLE2009-0144 Instituto de Salud Carlos III; PI 08/2028

The human amnion or amniotic membrane (HAM) has emerged as a novel and alternative source of stem cells. This tissue presents a reservoir of two types of stem cells from different embryological origins: human amniotic mesenchymal stem cells (hAMSCs), derived from the mesodermal germ layer, and human amniotic epithelial cells (hAECs), derived from the ectodermal germ layer. Both types of cells are different in disposition and morphology but have similar phenotypic characterization. hAMSCs are easily to isolate and can be expanded in vitro for several passages, obtaining a large amount of cells. Properties such as to be immune-privileged, antimicrobial, and antitumorogenic make themselves suitable for transplantation, cell therapy, and regenerative medicine. These cells were widely studied due to these properties and to their pluripotent capacity, being a promising clinically tool.

presentado en el 2014 World Congress on Osteoarthritis: Promoting Clinical and Basic Research in Osteoarthritis

The human amniotic membrane (HAM) is the innermost membrane surrounding the fetus. HAM is a highly abundant and readily available tissue that is becoming appreciated as an alternative to adult bone marrow mesenchymal stem cells (BM-MSCs) useful for cell therapy and regenerative medicine. This tissue provides high efficiency in noninvasive and safe MSC recovery with no intrusive procedures. HAM contains two cell types from different embryological origins: human amnion epithelial cells (hAECs), derived from the embryonic ectoderm, and human amnion mesenchymal stromal cells (hAMSCs), derived from the embryonic mesoderm. hAMSCs and hAECs are immune-privileged cells that can be isolated without the sacrifice of human embryos, avoiding immunological rejection problems and the ethical conflict of using human embryonic stem cells (hESCs). Regarding their immunophenotype, both cell types demonstrate the expression of the common well-defined human mesenchymal and embryonic stem cell markers and the absence of hematopoietic markers. Moreover, both cell populations have similar multipotential for in vitro differentiation into all three germ layers: ectoderm, mesoderm, and endoderm lineages. Indeed, the potential application of amnion-derived cells in a variety of diseases, in particular those associated with degenerative processes, is under clinical or preclinical investigation. The HAM has other biological properties important for tissue engineering, including anti-fibrosis, anti-inflammatory, anti-scarring, antimicrobial, as well as adequate mechanical properties and low immunogenicity. Therefore, amnion allografts are widely applied in ophthalmology, plastic surgery, dermatology, and gynecology. In this chapter, the localization, isolation, characterization, and differentiation potential of amnion-derived cells are discussed. Moreover, the potential clinical applications of either amnion-derived cells or the whole HAM are also reviewed.

Background. The interests in mesenchymal stem cells (MSCs) and their application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently hypoxia has been indicated as crucial for complete chondrogenesis. We aimed at analyzing bone marrow MSCs (BM-MSCs) differentiation capacity under normoxic and severe hypoxic culture conditions. Methods. MSCs were characterized by flow cytometry and differentiated towards adipocytes, osteoblasts, and chondrocytes under normoxic or severe hypoxic conditions. The differentiations were confirmed comparing each treated point with a control point made of cells grown in DMEM and fetal bovine serum (FBS). Results. BM-MSCs from the donors displayed only few phenotypical differences in surface antigens expressions. Analyzing marker genes expression levels of the treated cells compared to their control point for each lineage showed a good differentiation in normoxic conditions and the absence of this differentiation capacity in severe hypoxic cultures. Conclusions. In our experimental conditions, severe hypoxia affects the in vitro differentiation potential of BM-MSCs. Adipogenic, osteogenic, and chondrogenic differentiations are absent in severe hypoxic conditions. Our work underlines that severe hypoxia slows cell differentiation by means of molecular mechanisms since a decrease in the expression of adipocyte-, osteoblast-, and chondrocyte-specific genes was observed. This study was supported by Grants from Servizo Galego de Saúde, Xunta de Galicia (PS07/84), Cátedra Bioiberica de la Universidade da Coruña and Instituto de Salud Carlos III CIBER BBN CB06-01-0040, Ministerio Ciencia e Innovacion PLE2009-0144 and Fondo Investigacion Sanitaria-PI 08/2028 with participation of funds from FEDER (European Community); Tamara Hermida-Gómez is the beneficiary of a Contract from Fondo de Investigación Sanitaria (2008), Spain. Emma Muiños-López is supported by the Rheumatology Spanish Foundation, Spain SI

Objectives:To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies.Materials and Methodology:The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed.Results:Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046vs0.028, respectively, pCCND1mRNA and protein expression levels, and immunopositivity forKi67revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higherSOX9andCol IIexpression in chondrocytes from deep than from superficial zone (pTstudent test).Conclusions:The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes.

[Abstract] We aim to estimate the diagnostic performances of anterior gradient homolog-2 (AGR2) and Leucine-rich repeat-containing-G-protein-coupled receptor 5 (LGR5) in peripheral blood (PB) as mRNA biomarkers in colorectal cancer (CRC) and to explore their prognostic significance. Real-time PCR was used to analyze AGR2 and LGR5 in 54 stages I-IV CRC patients and 19 controls. Both mRNAs were significantly increased in PB from CRC patients compared to controls. The area under the receiver-operating characteristic curves were 0.722 (p = 0.006), 0.376 (p = 0.123) and 0.767 (p = 0.001) for AGR2, LGR5 and combined AGR2/LGR5, respectively. The AGR2/LGR5 assay resulted in 67.4% sensitivity and 94.7% specificity. AGR2 correlated with pT3–pT4 and high-grade tumors. LGR5 correlated with metastasis, R2 resections and high-grade. The progression-free survival (PFS) of patients with high AGR2 was reduced (p = 0.037; HR, 2.32), also in the stage I-III subgroup (p = 0.046). LGR5 indicated a poor prognosis regarding both PFS (p = 0.007; HR, 1.013) and overall survival (p = 0.045; HR, 1.01). High AGR2/LGR5 was associated with poor PFS (p = 0.014; HR, 2.8) by multivariate analysis. Our findings indicate that the assessment of AGR2 and LGR5 in PB might reflect the presence of circulating tumor cells (CTC) and stem cell like CTC in CRC. Increased AGR2 and LGR5 are associated with poor outcomes. Rede Galega de Investigación sobre Cancro Colorrectal; grant 5090252501

Stem cells within many tissues are thought to reside within a niche formed by a group of surrounding cells and their extracellular matrices, which provide an optimal microenvironment for the stem cells to function. In general, the niche is thought to consist of a highly organized microenvironment in which various factors, such as signals coming from secreted cytokines, extracellular matrix interactions, and intercellular adhesion, are thought to work cooperatively to maintain the undifferentiated stem cell phenotype. Among stem cells, adult stem cells are often localized into specific niches where they utilize many, but not necessarily all, of the external and intrinsic factors used by the embryonic counterparts in selecting a specific fate. Within the niche, stem cells are able to maintain their ability for selfrenewal as well as their potential so that, consequently, detachment from the niche compartment induces stem cell differentiation and loss of self-renewal. Thus, when a stem cell begins to divide, it is thought that one daughter cell remains into niche to replace the original stem cell whereas other daughter cell is expelled out of niche and starts its process of differentiation. In this process, a cell retains self-renewal and differentiation inhibitory factors, so that keep being stem cell, whereas another daughter cell is destined to proliferate during a certain number of divisions for finally differentiate along a particular lineage. This latter daughter cell will receive too few stemness factors to maintain as stem cell, and/or inherit proliferation and/or differentiation factors that can overcome its stem cell phenotype. To maintain tissue homeostasis and correct functioning of organism, the number of daughter cells that retain stem cell identity must be strictly controlled such that differentiated cells can be generated in response to any injury. Likewise, the rate of division of stem cells into niche must be tightly controlled since an overproduction of daughter cells destined to be differentiated may be harmful because may result in cancer generation. In the present chapter, we speculate cancer stem cell niche for as well as the mechanisms that influence on the generation of daughter cells.

The hedgehog (Hh) pathway is one of the fundamental signal transduction pathways in animal development and is also involved in stem-cell maintenance and carcinogenesis. The hedgehog (hh) gene was first discovered in Drosophila, and members of the family have since been found in most metazoan. Hh proteins are composed of two domains, an aminoterminal domain HhN, which has the biological signal activity, and a carboxy-terminal autocatalytic domain HhC, which cleaves Hh into two parts in an intramolecular reaction and adds a cholesterol moiety to HhN. HhC has a sequence similarity to the self-splicing inteins, and the shared region is termed Hint. HhN is modified by cholesterol at its carboxyl terminus and by palmitate at its amino terminus in both flies and mammals. The modified HhN is released from the cell and travels through the extracellular space. On binding its receptor Patched, it relieves the inhibition that Patched exerts on Smoothened, a G-proteincoupled receptor. The resulting signalling cascade converges on the transcription factor Cubitus interruptus (Ci), or its mammalian counterparts, the Gli proteins, which activate or repress target genes. The Hh family of morphogens plays important instructional roles in the development of numerous metazoan structures (Ingham & McMahon, 2001). The Hh ligands, Sonic, Indian and Desert Hh in vertebrates and Hh in Drosophila, signal through binding to the membrane receptor Patched (Ptc) (Chen & Struhl, 1996), to reverse the Ptcmediated inhibition of signalling by the trans-membrane protein Smoothened (Smo) (Alcedo et al., 1996). This allows Smo to activate the intracellular signalling components, resulting in stabilization of down-stream transcriptional activator(s) and activation of target genes (Hooper & Scott, 1989). Transcription activation is facilited through the Gli family of transcription factors in vertebrates (Ingham & McMahon, 2001). Hh signalling can initiate cellular growth, division, lineage specification, axon guidance and function as a survival factor (Cohen, 2009). Given this range of biological functions, it is not surprising that mutations in components of the Hh pathway are associated with both developmental defects and tumor progression (Cohen, 2009). Disruption of PTC, which functions as a negative regulator of the pathway, is implicated in cancer development in both inherited and sporadic cancers. Mutations in PTC and/or SMO trigger inappropriate activation of the Hh pathway, and have been identified in tumor types including basal cell carcinoma

[Abstract] Objective: The purposes of this project were to quantify the cells expressing the mesenchymal stem cell (MSC) marker CD271 in synovial membranes from human osteoarthritic (OA) and healthy joints, and to determine if those CD271 cells were involved in spontaneous human cartilage repair and were beneficial for the repair of human articular cartilage defects. Methods: The coexpression of CD44/CD271, CD90/CD271, and CD105/CD271 antigens was determined by immunofluorescence in OA and healthy synovial membranes and during spontaneous cartilage repair. Isolated MSCs from the bone marrow of four OA patients (mean age: 64 years) were magnetically separated into MSC CD271+ and MSC CD271 subsets. The separated cell subsets were then implanted into 2 mm focal defects of articular cartilage. These implants were cultured in chondrogenic differentiation medium supplemented with recombinant human transforming growth factor-beta3 for 8 weeks. The repair tissues were analyzed by histochemistry (hematoxylin–eosin and safranin O) and immunohistochemistry for collage types I and II. Results: Cells expressing the CD271 antigen were diffusely distributed in OA synovial membranes and localized in the subintimal zone in healthy synovial membranes. The number of cells expressing MSC markers was higher in OA synovial membranes than in synovia from healthy joints, corresponding to the highest level of coexpression of CD90/CD271 antigens (9.8% vs. 2.6%). Spontaneous repair tissue showed more cells expressing the CD271 antigen (9.9% ± 4.0%). The highest levels of expression were found to be associated with CD44; 64% of positive CD271 cells coexpressed the CD44 antigen. In both implant cell types, the repair tissue morphology resembled articular cartilage, having an extracellular matrix with a hyaline aspect and numerous lacunae containing cells, and was immunopositive for collagen types I and II. Statistical analyses of the repair tissue demonstrated that the implantation of MSC CD271+ provided such benefits as a greater filling of the chondral defect and better integration between the repair tissue and native cartilage. Safranin O staining of repair tissue was negative in implants of MSC CD271- but more positive in implants with MSC CD271+. The overall histologic score for CD271 implants was 9.5 ± 0.89 and 12.19 ± 1.01 for CD271+ implants. Conclusions: Synovial membranes from OA patients contain more cells expressing CD271 antigen than those from healthy joints, and spontaneous cartilage repair tissue contains cells positive for CD271 antigen. These data suggest the involvement of CD271 antigen in spontaneous cartilage repair and indicate that the cell subset MSC CD271+ provides higher quality chondral repair than the CD271- subset. Galicia. Consellería de Economía, Industria e Comercio; PGIDIT05SAN08PR Galicia. Consellería de Economía, Industria e Comercio; PGIDIT04PXIC91602PN Galicia. Consellería de Economía, Industria e Comercio; PGIDIT04PXIC91601PN Instituto de Salud Carlos III; FISCP03/00127 Instituto de Salud Carlos III; FIS05/2495

Objective.To quantify cells expressing mesenchymal stem cell (MSC) markers in synovial membranes from human osteoarthritic (OA) and healthy joints.Methods.Synovial membranes from OA and healthy joints were digested with collagenase and the isolated cells were cultured. Synovial membrane-derived cells were phenotypically characterized for differentiation experiments using flow cytometry to detect the expression of mesenchymal markers (CD29, CD44, CD73, CD90, CD105, CD117, CD166, and STRO-1) and hematopoietic markers (CD34 and CD45). Chondrogenesis was assessed by staining for proteoglycans and collagen type II, adipogenesis by using a stain for lipids, and osteogenesis by detecting calcium deposits. Coexpression of CD44, CD73, CD90, and CD105 was determined using immunofluorescence.Results.Cells expressing MSC markers were diffusely distributed in OA synovial membranes; in healthy synovial membrane these cells were localized in the subintimal zone. More numerous MSC markers in OA synovial membranes were observed in cells also expressing the CD90 antigen. FACS analysis showed that more than 90% of OA synovial membrane-derived cells were positive for CD44, CD73, and CD90, and negative for CD34 and CD45. OA synovial membrane-derived cells were also positive for CD29 (85.23%), CD117 (72.35%), CD105 (45.5%), and STRO-1 (49.46%). Micropellet analyses showed that the culture of cells with transforming growth factor-ß3 stimulated proteoglycan and collagen type II synthesis.Conclusion.Synovial membranes from patients with OA contain more cells positive for CD44, CD90, and CD105 antigens than those from joints with undamaged cartilage.

[Abstract] The human amniotic membrane (HAM) is a highly abundant and readily available tissue. This amniotic tissue has considerable advantageous characteristics to be considered as an attractive material in the field of regenerative medicine. It has low immunogenicity, anti-inflammatory properties and their cells can be isolated without the sacrifice of human embryos. Since it is discarded post-partum it may be useful for regenerative medicine and cell therapy. Amniotic membranes have already been used extensively as biologic dressings in ophthalmic, abdominal and plastic surgery. HAM contains two cell types, from different embryological origins, which display some characteristic properties of stem cells. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. Both populations have similar immunophenotype and multipotential for in vitro differentiation into the major mesodermal lineages, however they differ in cell yield. Therefore, HAM has been proposed as a good candidate to be used in cell therapy or regenerative medicine to treat damaged or diseased tissues. Servizo Galego de Saúde; PS07/84 Instituto de Salud Carlos III; CIBER BBNCB06-01-0040 Instituto de Salud Carlos III; PI 08/2028 Ministerio Ciencia e Innovacion; PLE2009-0144

[Abstract] Introduction: The human amniotic membrane is a highly abundant and readily available tissue that may be useful for regenerative medicine and cell therapy. Aim: To compare two previously published protocols for the isolation of human amnion mesenchymal stromal cells (hAMSCs), including their phenotypic characterization and in vitro potential for differentiation toward osteogenic, adipogenic, and chondrogenic mesodermal lineages. Materials and Methods: Human placentas were obtained from selected caesarean-sectioned births. Two different protocols (Alviano et al.1 and Soncini et al.2) for the isolation of hAMSCs were performed. After monolayer expansion of adherent cells from both protocols, the cells were characterized by flow cytometry and for multi- potentiality, as assessed by their capability to differentiate toward adipocyte-, osteoblast-, and chondrocyte-like cells. Results: Both protocols yielded hAMSCs that showed plastic adherence, fibroblast-like growth, and well-defined human MSC markers. The cell yield and mesodermal differentiation capability of hAMSCs were higher in cells isolated using the Soncini protocol. Conclusions: Our data demonstrated the successful isolation of hAMSCs from full-term placentas using two published protocols. Differences between the two protocols in cell yield and in vitro differentiation potential are shown. Servizo Galego de Saúde; PS07/84 Instituto de Salud Carlos III; CIBER BBN CB06-01-0040