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Osteoarthritis (OA) is a heterogeneous disease in which the cross-talk between the cells from different tissues within the joint is affected as the disease progresses. Extracellular vesicles (EVs) are known to have a crucial role in cell-cell communication by means of cargo transfer. Subchondral bone (SB) resident cells and its microenvironment are increasingly recognised to have a major role in OA pathogenesis. The aim of this study was to investigate the EV production from OA SB mesenchymal stromal cells (MSCs) and their possible influence on OA chondrocytes. Small EVs were isolated from OA-MSCs, characterized and cocultured with chondrocytes for viability and gene expression analysis, and compared to small EVs from MSCs of healthy donors (H-EVs). OA-EVs enhanced viability of chondrocytes and the expression of chondrogenesis-related genes, although the effect was marginally lower compared to that of the H-EVs. miRNA profiling followed by unsupervised hierarchical clustering analysis revealed distinct microRNA sets in OA-EVs as compared to their parental MSCs or H-EVs. Pathway analysis of OA-EV miRNAs showed the enrichment of miRNAs implicated in chondrogenesis, stem cells, or other pathways related to cartilage and OA. In conclusion, OA SB MSCs were capable of producing EVs that could support chondrocyte viability and chondrogenic gene expression and contained microRNAs implicated in chondrogenesis support. These EVs could therefore mediate the cross-talk between the SB and cartilage in OA potentially modulating chondrocyte viability and endogenous cartilage regeneration.

[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

[Abstract] Background: The biological mechanisms that contribute to atrophic long bone non-union are poorly understood. Multipotential mesenchymal stromal cells (MSCs) are key contributors to bone formation and are recognised as important mediators of blood vessel formation. This study examines the role of MSCs in tissue formation at the site of atrophic non-union. Materials and Methods: Tissue and MSCs from non-union sites (n = 20) and induced periosteal (IP) membrane formed following the Masquelet bone reconstruction technique (n = 15) or bone marrow (n = 8) were compared. MSC content, differentiation, and influence on angiogenesis were measured in vitro. Cell content and vasculature measurements were performed by flow cytometry and histology, and gene expression was measured by quantitative polymerase chain reaction (qPCR). Results: MSCs from non-union sites had comparable differentiation potential to bone marrow MSCs. Compared with induced periosteum, non-union tissue contained similar proportion of colony-forming cells, but a greater proportion of pericytes (p = 0.036), and endothelial cells (p = 0.016) and blood vessels were more numerous (p = 0.001) with smaller luminal diameter (p = 0.046). MSCs showed marked differences in angiogenic transcripts depending on the source, and those from induced periosteum, but not non-union tissue, inhibited early stages of in vitro angiogenesis. Conclusions: In vitro, non-union site derived MSCs have no impairment of differentiation capacity, but they differ from IP-derived MSCs in mediating angiogenesis. Local MSCs may thus be strongly implicated in the formation of the immature vascular network at the non-union site. Attention should be given to their angiogenic support profile when selecting MSCs for regenerative therapy.

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] 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

Osteoarthritis (OA) is the most common degenerative joint disorder. Multipotential stromal cells (MSCs) have a crucial role in joint repair, but how OA severity affects their characteristics remains unknown. Knee OA provides a good model to study this, as osteochondral damage is commonly more severe in the medial weight-bearing compartment compared to lateral side of the joint. This study utilised in vitro functional assays, cell sorting, gene expression and immunohistochemistry to compare MSCs from medial and lateral OA femoral condyles. Despite greater cartilage loss and bone sclerosis in medial condyles, there was no significant differences in MSC numbers, growth rates or surface phenotype. Culture-expanded and freshly-purified medial-condyle MSCs expressed higher levels of several ossification-related genes. Using CD271-staining to identify MSCs, their presence and co-localisation with TRAP-positive chondroclasts was noted in the vascular channels breaching the osteochondral junction in lateral condyles. In medial condyles, MSCs were additionally found in small cavities within the sclerotic plate. These data indicate subchondral MSCs may be involved in OA progression by participating in cartilage destruction, calcification and sclerotic plate formation and that they remain abundant in severe disease. Biological or biomechanical modulation of these MSCs may be a new strategy towards cartilage and bone restoration in knee OA.

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

To date, the therapeutic efficacy of human mesenchymal stem cells (hMSCs) has been investigated in various clinical trials with moderate or in some cases inconsistent results. The still elusive reproducibility relates in part with constitutive differences in the cell preparation, translated into variable “cell potencies”. Other factors include poor cell homing and survival, and age/disease-associated host tissue impairment. It is well accepted that within in vivo niches MSCs exist as heterogeneous cell populations with different stemness propensities and supportive functions. Phenotype-based MSC purification of homogeneous subsets can result in cell populations with distinct biological functions. In addition, preclinical studies have shown that MSC functionalization in vitro, via cell priming, can boost their immunomodulatory, trophic and reparative capacities in vivo. Therefore, in the present review we discuss how phenotype-based MSC purification and MSC priming technologies can contribute to an improved MSC-based product for safer and more effective therapeutic applications.

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

Large bone defects are ideally treated with autografts, which have many limitations. Therefore, osteoconductive scaffolds loaded with autologous bone marrow (BM) aspirate are increasingly used as alternatives. The purpose of this study was to compare the growth of multipotential stromal cells (MSCs) from unprocessed BM on a collagen‐containing bovine bone scaffold (Orthoss® Collagen) with a non‐collagen‐containing bovine bone scaffold, Orthoss®. Another collagen‐containing synthetic scaffold, Vitoss® was included in the comparison. Colonization of scaffolds by BM MSCs (n = 23 donors) was evaluated using microscopy, colony forming unit‐fibroblast assay and flow‐cytometry. The number of BM MSCs initially attached to Orthoss® Collagen and Vitoss® was similar but greater than Orthoss® (p = 0.001 and p = 0.041, respectively). Furthermore, the number of MSCs released from Orthoss® Collagen and Vitoss® after 2‐week culture was also higher compared to Orthoss® (p = 0.010 and p = 0.023, respectively). Interestingly, collagen‐containing scaffolds accommodated larger numbers of lymphocytic and myelomonocytic cells. Additionally, the proliferation of culture‐expanded MSCs on Orthoss® collagen and Vitoss® was greater compared to Orthoss® (p = 0.047 and p = 0.004, respectively). Collectively, collagen‐containing scaffolds were superior in supporting the attachment and proliferation of MSCs when they were loaded with unprocessed BM aspirates. This highlights the benefit of collagen incorporation into bone scaffolds for use with autologous bone marrow aspirates as autograft substitutes. © 2015 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:597–606, 2016.

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.

[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.

Human Cartilage Tissue Engineering Using Type I Collagen/Heparan Sulfate Scaffolds Introduction: Cartilage engineering may provide a promising alternative solution to current methods of cartilage repair. The aim of this study was to evaluate the suitability of type I collagen (Col I) scaffolds with and without heparan sulfate (HS) to support attachment, proliferation and chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Materials and Methods: hBMSCs were cultured in Col I, Col I+1%HS, Col I+2%HS and Col I+3%HS scaffolds in both chondrogenic and non-differentiation media for 15 and 30 days. The resulting neo-tissues were analyzed using histochemistry, immunohistochemistry, electron microscopy (EM) and molecular biology. Collagen released into the media by the constructs was also measured by dye-binding assays. Results: Our results showed successful growth and proliferation of hBMSCs on all scaffolds analyzed. Better results were obtained in chondrogenic cultures after 30 days, in which we observed oval/ rounded cells along the scaffolds, and extracellular matrix (ECM) by EM; this ECM was strongly positive for proteoglycan (PG) safranin O staining and staining for type II (Col II). Greater total collagen release was found in supernatants of chondrogenic cultures than in controls. The best results for all analyses were found in scaffolds including HS. Conclusions: We conclude that the Col I and HS scaffolds used in this study are suitable supports for hBMSCs to differentiate toward chondrocyte-like cells in chondrogenic medium. We observe that the addition of HS to Col I scaffolds improves the chondrogenic phenotype of the cells, with Col I+3%HS being the best scaffold.

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

(score decline -64%) and biochemical analysis of GAG (-75%) and total collagen (-30%) content. Gene expression of catabolic enzymes (MMP13, MMP1, MMP2), and genes involved in inflammatory response (IL8, CCL2, CCL4 CXCL2) was significantly increased (>4-fold) and genes related to ECM formation (COL2A1, COMP, Aggrecan, COL8A1, COL11A1) were decreased (

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