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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] 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 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.

[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