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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

[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.

[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

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

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.

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 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

[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

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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