Retinitis pigmentosa (RP), a genetically heterogeneous group of diseases together with age-related macular degeneration (AMD), are the leading causes of permanent blindness and are characterized by the progressive dysfunction and death of the light sensing photoreceptors of the retina. Due to the limited regeneration capacity of the mammalian retina, the scientific community has invested significantly in trying to obtain retinal progenitor cells from embryonic stem cells (ESC). These represent an unlimited source of retinal cells, but it has not yet been possible to achieve specific populations, such as photoreceptors, efficiently enough to allow them to be used safely in the future as cell therapy of RP or AMD. In this study, we generated a high yield of photoreceptors from directed differentiation of mouse ESC (mESC) by recapitulating crucial phases of retinal development. We present a new protocol of differentiation, involving hypoxia and taking into account extrinsic and intrinsic cues. These include niche-specific conditions as well as the manipulation of the signaling pathways involved in retinal development. Our results show that hypoxia promotes and improves the differentiation of mESC toward photoreceptors. Different populations of retinal cells are increased in number under the hypoxic conditions applied, such as Crx-positive cells, S-Opsin-positive cells, and double positive cells for Rhodopsin and Recoverin, as shown by immunofluorescence analysis. For the first time, this manuscript reports the high efficiency of differentiation in vivo and the expression of mature rod photoreceptor markers in a large number of differentiated cells, transplanted in the subretinal space of wild-type mice.

The existing in vitro and in vivo models for studying osteoarthritis have significant limitations in replicating the complexity of joint tissues. This research aims to validate a Tissue-On-a-Chip system for osteoarthritis research. Osteochondral tissues obtained from knee replacement surgeries of patients with osteoarthritis were cultured in an Organ-On-a-Chip system. This system was designed to supply oxygen and glucose to the cartilage from the bone. The distribution of oxygen and glucose was evaluated by fluorescence using Image-iT Green Hypoxia and 2-NBDG, respectively. Cytotoxicity was measured using lactate dehydrogenase (LDH) levels in chip cultures compared to plate cultures (12 tissues per method). Glycosaminoglycans (GAGs), alkaline phosphatase (ALP), Coll2-1, and procollagen type II N-terminal propeptide (PIINP) were measured in the perfused medium of the Tissue-On-a-Chip over a period of 70 days. Fluorescence of Image-iT Green Hypoxia was observed only in the cartilage area, while 2-NBDG was distributed throughout the tissue. An increase in LDH levels was noted in the plate cultures on day 24 and in the Tissue-On-a-Chip cultures on day 63. Compared to the start of the culture, GAG content increased on day 52, while ALP showed variations. A notable increase in GAG, ALP, and Coll2-1 levels was observed on day 59. PIINP levels remained stable throughout the experiment. The validated osteochondral Tissue-On-a-Chip system can replicate the joint microenvironment, with hypoxic conditions in cartilage and normoxic conditions in bone. Tissue survival and component stability were maintained for approximately two months. This platform is a useful tool for evaluating new drugs and represents a viable alternative to animal models.

In this study of the alterations of Glypicans 1 to 6 (GPCs) and Notum in plasma, bone marrow mesenchymal stromal cells (BM-MSCs) and osteoblasts in Osteoarthritis (OA), the levels of GPCs and Notum in the plasma of 25 patients and 24 healthy subjects were measured. In addition, BM-MSCs from eight OA patients and eight healthy donors were cultured over a period of 21 days using both a culture medium and an osteogenic medium. Protein and gene expression levels of GPCs and Notum were determined using ELISA and qPCR at 0, 7, 14 and 21 days. GPC5 and Notum levels decreased in the plasma of OA patients, while the BM-MSCs of OA patients showed downexpression of GPC6 and upregulation of Notum. A decrease in GPC5 and Notum proteins and an increase in GPC3 were found. During osteogenic differentiation, elevated GPCs 2, 4, 5, 6 and Notum mRNA levels and decreased GPC3 were observed in patients with OA. Furthermore, the protein levels of GPC2, GPC5 and Notum decreased, while the levels of GPC3 increased. Glypicans and Notum were altered in BM-MSCs and during osteogenic differentiation from patients with OA. The alterations found point to GPC5 and Notum as new candidate biomarkers of OA pathology.

Background Osteoarthritis (OA) is accompanied by an excessive formation of underlying bone caused by homeostatic alterations. It has been described that in OA joints there is an up-regulation of the WNT/β-catenine pathway driving to the differentiation of Mesenchymal Stromal Cells (MSCs) into osteocytes [1]. Glypicans 1-6 act as co-receptors of WNT pathway, improving interactions between molecules [2]. On the other hand, NOTUM plays a role as a negative regulator of the pathway preventing WNT binding to its receptors and favouring the soluble form of Glypicans [3]. All these evidences lead us to think about the importance of Glypicans 1-6 and NOTUM in the osteogenic differentiation of MSCs in OA disease. Objectives To determine the differences at gene and protein levels of Glypicans 1-6 and NOTUM in MSCs between OA patients and controls at baseline and during induced in vitro osteogenic differentiation. Methods Bone Marrow Mesenchymal Stromal Cells (BM-MSCs) from both, 8 OA patients and 8 healthy donors were isolated from BM samples. BM samples were obtained by joint replacement and traumatic fractures. BM-MSCs were cultured during 21 days with normal culture medium and with osteogenic inducing medium. Cells and supernatant were recovered at 1, 7, 14 and 21 days. Protein levels were determined by ELISA in cells supernatant. Extraction and purification of RNA from cells and cDNA synthesis were performed. Glypicans 1-6 and NOTUM gene-expression was analysed by qPCR and normalized with housekeeping genes β-actin and RNA18S. The data were calculated with the method of fold change (2^-ΔΔCt). Statical analysis were performed with GraphPad Prism 8.0. For the detection of outliers, the ROUT method (Q=1%) was used. Data were analyzed using t-test. Results The mRNA levels of cells with normal culture medium were up-regulated in GPC6 (t=7d; p=0,016) in OA patients. During osteogenic differentiation, was observed a statistically significant up-regulation in levels of OA patients vs controls of GPC2 (t=14d; p=0,034), GPC4 (t=14d, p=0,025; t=21d, p=0,017), GPC5 (t=14d, p=0,007), GPC6 (t=14d, p=0,013) and NOTUM (t=14d, p=0,009) and down-regulation of GPC3 (t=7d, p=0,034). In addition, protein levels of NOTUM were lower compared to the control group in OA BM-MSCs (t=1d, p=0,049) and in the differentiation to osteocytes (t=1d, p=0,049; t=7d, p=0,043; t=14d, p=0,016). GPC3 levels were higher OA patients in all the times and in the two conditions (p < 0,0001). In the differentiation, GPC2 were lower (t=7d, p< 0,0001). GPC1, GPC4 and GPC6 were not present in any case. Conclusion Our results evidence a dysregulation in the glypicans in BM-MSCs of OA patients and, specially, during osteogenic differentiation. NOTUM, extracellular negative regulator of the WNT/β-catenine signaling pathway, is decreased at protein level in OA patients, despite the fact that gene expression is elevated. Thus, our data confirm the differences in expression in NOTUM and Glypicans between OA patients and healthy controls. Further studies are needed proposing these molecules, specially NOTUM, as an effective treatment of the disease. References [1]Tornero-Esteban et al., 2015 “Altered Expression” PLoS One. [2]Boudin et al., 2013 “The role” Semin Arthritis Rheum. [3]Nusse R., 2015 “Cell signalling” Nature. Acknowledgements: NIL. Disclosure of Interests None Declared.

Study of alterations in Osteoarthritis (OA) of Glypicans 1 to 6 (GPCs) and Notum in plasma, bone marrow mesenchymal stromal cells (BM-MSCs) and osteoblasts. Plasma levels of GPCs and Notum were measured of 25 patients and 24 healthy subjects. In addition, BM-MSCs from eight OA patients and eight healthy donors were cultured during 21 days with culture me-dium and osteogenic medium. Protein and gene expression levels of GPCs and Notum were de-termined by ELISA and qPCR at 0, 7 ,14 and 21 days. GPC5 and Notum have been found de-creased in OA patients’ plasma. OA BM-MSCs showed downexpression of GPC6 and upregula-tion of Notum. A decrease in GPC5 and Notum proteins and an increase in GPC3 were found. During osteogenic differentiation, elevated GPC2, 4, 5, 6 and Notum mRNA levels and decreased GPC3 were observed in OA. Furthermore, the protein levels of GPC2, GPC5 and Notum de-creased and GPC3 increased. Glypicans and Notum are altered in BM-MSCs and during osteo-genic differentiation from patients with OA. Furthermore, GPC5 and Notum can be proposed as new biomarkers of the pathology. The alterations found indicate that Notum could be a prom-ising therapeutic strategy for Osteoarthritis.

Osteoarthritis (OA) is the most common multifactorial joint disease characterized by progressive cartilage degradation and impaired tissue repair. Osteochondral defects represent a major clinical challenge within OA, as damage to cartilage and underlying bone can initiate degenerative changes and contribute to joint deterioration. The Wnt/β-catenin signaling pathway plays an important role in OA pathogenesis, and its dysregulation contributes to chondrocyte catabolism and cartilage loss. NOTUM, an extracellular Wnt inhibitor, has emerged as a potential therapeutic modulator capable of restoring signaling balance and promoting cartilage homeostasis. This study aimed to evaluate the efficacy of NOTUM compared with hyaluronic acid (HA), human adipose-derived mesenchymal stromal cells (hAd-MSCs), and Colchicine in a rabbit osteochondral defect model relevant to osteoarthritis. Twenty-seven New Zealand White rabbits underwent standardized femoral condyle injury and received single-dose treatments. Serum levels of cartilage biomarkers—Procollagen Type IIA N-terminal Propeptide (PIIANP) and Cartilage Oligomeric Matrix Protein (COMP)—were measured by ELISA at 4, 6, and 8 weeks post-surgery, and histological repair at week 12 was assessed using the modified O’Driscoll scoring system. NOTUM treatment significantly increased PIIANP and decreased COMP levels compared with HA, indicating enhanced cartilage synthesis and reduced degradation. Histological scores confirmed superior surface morphology and tissue composition in NOTUM-treated joints. These findings suggest that NOTUM performs a protective and regenerative effect through Wnt/β-catenin modulation, supporting the conclusion that it enhances osteochondral defect repair and motivating further studies of NOTUM as an OA therapy.