Immortalization of human mesenchymal stem cells with SV40 T antigen

Las células madre mesenquimales (CMMs) son células madre adultas multipotentes con una alta capacidad de proliferación, cultivables in vitro y que presentan propiedades inmunomoduladoras y antiinflamatorias. Es por ello que tienen un gran interés en investigación y en terapia celular para el estudio y el tratamiento de enfermedades crónicas en las que se requiere la regeneración de tejidos, como es el caso de la artrosis, una enfermedad degenerativa de las articulaciones que afecta al 20% de la población mundial y al 70% de los mayores de 60 años, para la que actualmente no existe ningún tratamiento capaz de regenerar el tejido dañado. Sin embargo, para estas aplicaciones es necesario expandir las CMMs in vitro, y este proceso puede acabar provocando la senescencia de las células y la adquisición de alteraciones relacionadas con la misma. Una forma de evitar la senescencia de las células humanas en cultivo es obtener una línea celular con un fenotipo estable y una capacidad de proliferación ilimitada mediante la inmortalización de las células por transfección de genes virales, como el antígeno T del virus SV40 (SV40 LT), o humanos, como el gen de la transcriptasa reversa de la telomerasa humana (hTERT). Por estos motivos se planteó el objetivo de este proyecto de investigación, que fue la obtención de líneas celulares para el estudio de la artrosis mediante la inmortalización de CMMs humanas procedentes de la médula ósea (CMMs-MO) de un paciente con artrosis y un donante sano. Aunque no se ha conseguido la generación de una línea celular, con el protocolo de inmortalización empleado se han obtenido algunos resultados prometedores que constituyen la base para continuar esta investigación, ya que se ha conseguido generar retrovirus portadores de la región codificante temprana del virus SV40 y transfectar de forma eficiente CMMs-MO de dos pacientes con artrosis y un donante sano con estos retrovirus.Traballo fin de mestrado (UDC.CIE). Bioloxía molecular, celular e xenética. Curso 2015/201

Generation and Characterization of Mesenchymal Cell Lines for Osteorchondral Regeneration Research

Programa Oficial de Doutoramento en Ciencias da Saúde. 5007V01[Resumo] A rexeneración do óso e da cartilaxe tras sufrir un traumatismo ou unha enfermidade dexenerativa segue sendo un gran desafío clínico. Debido á súa capacidade de auto-renovación e multi-diferenciación, as células mesenquimais estromais (MSC) son unha fonte celular moi prometedora para a rexeneración destes tecidos, pero a investigación neste campo está limitada pola tendencia das MSC á senescencia ao seren expandidas en cultivo. A inmortalización das MSC permítelles superar a senescencia, o que supón un impulso para os avances na investigación. Neste estudo desenvolveuse un método para inmortalizar MSC derivadas de doantes de idade avanzada mediante inoculación centrífuga de dous xenes de inmortalización: o antíxeno T grande do virus de simio 40 (SV40LT) e a transcriptase reversa da telomerase humana (hTERT). As MSC inmortalizadas son fenotipicamente similares ás MSC primarias e son capaces de diferenciarse cara ás tres liñaxes esqueléticas, aínda que se inclinan cara á ruta de diferenciación osteoxénica. Os condrocitos articulares e os sinoviocitos pódense inmortalizar empregando o mesmo método, pero os condrocitos inmortalizados son metabolicamente diferentes dos condrocitos articulares primarios. Estas células poden ser útiles como parte de modelos in vitro de rexeneración dos tecidos articulares ou de enfermidade osteocondral.[Resumen] La regeneración del hueso y el cartílago tras sufrir un traumatismo o una enfermedad degenerativa continúa siendo un gran desafío clínico. Debido a su capacidad de auto-renovación y multi-diferenciación, las células mesenquimales estromales (MSC) son una fuente celular prometedora para la regeneración de estos tejidos, pero la investigación en este campo se ve limitada por la tendencia de las MSC a la senescencia en cultivo. La inmortalización de las MSC les permite superar la senescencia, impulsando así los avances en la investigación. En este estudio, se ha desarrollado un método para inmortalizar MSC derivadas de donantes de edad avanzada mediante inoculación centrífuga de dos genes de inmortalización: el antígeno T grande del virus de simio 40 (SV40LT) y la transcriptasa reversa de la telomerasa humana (hTERT). Las MSC inmortalizadas son fenotípicamente similares a las MSC primarias y son capaces de diferenciarse hacia los tres linajes esqueléticos, aunque tienen tendencia a seguir la ruta de diferenciación osteogénica. Los condrocitos articulares y los sinoviocitos se pueden inmortalizar utilizando el mismo método, pero los condrocitos inmortalizados son metabólicamente diferentes de los condrocitos articulares primarios. Estas células pueden ser útiles como parte de modelos in vitro de regeneración de los tejidos articulares o de enfermedad osteocondral.[Abstract] Regeneration of bone and cartilage after trauma or age-related degenerative diseases remains a major clinical challenge. Due to their self-renewal and multi-differentiation potential, mesenchymal stromal cells (MSCs) are a promising cell source for bone and cartilage regeneration, but research on this field is impaired by MSCs’ predisposition to senescence when culture-expanded. Immortalization of MSCs allows them to bypass senescence, thus boosting the advances in MSC research. In this study, a method has been developed to immortalize MSCs derived from elderly donors by spinoculation of two immortalization genes: simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). Immortalized MSCs are phenotypically similar to primary MSCs and are able to differentiate to the three skeletal lineages, although their multi-differentiation potential is unbalanced towards the osteogenic pathway. Articular chondrocytes and synoviocytes can also be immortalized by the same method, but immortalized chondrocytes are metabolically different from primary articular chondrocytes. These immortalized cells can be useful as part of in vitro models of osteochondral regeneration and disease

Current Development of Alternative Treatments for Endothelial Decompensation: Cell-Based Therapy

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; R2016/036Xunta de Galicia; ED431B 2020/55Xunta de Galicia; ED481B 2017/029Xunta de Galicia; ED481A-2019/206Xunta de Galicia; ED481A-2017/280This 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

Generation of a human control iPS cell line (ESi080‐A) from a donor with no rheumatic diseases

[Abstract] 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.Instituto de Salud Carlos III; PI17/0219

Versatility of Induced Pluripotent Stem Cells (iPSCs) for Improving the Knowledge on Musculoskeletal Diseases

[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; PI17/02197Xunta de Galicia; R2016/036Xunta de Galicia; R2014/050Xunta de Galicia; CN2012/142Xunta de Galicia; GPC2014/04

Usefulness of mesenchymal cell lines for bone and cartilage regeneration research

[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; R2016/036Deputación da Coruña; BINV-CS/2016Xunta de Galicia; R2014/050Xunta de Galicia; CN2012/142Xunta de Galicia; GPC2014/04

Reduced Levels of H₂S in Diabetes-Associated Osteoarthritis Are Linked to Hyperglycaemia, Nrf-2/HO-1 Signalling Downregulation and Chondrocyte Dysfunction

[Abstract] Different findings indicate that type 2 diabetes is an independent risk factor for osteoarthritis (OA). However, the mechanisms underlying the connection between both diseases remain unclear. Changes in the balance of hydrogen sulphide (H₂S) are thought to play an important role in the pathogenesis of diabetes and its complications, although its role is still controversial. In this study, we examined the modulation of H₂S levels in serum and chondrocytes from OA diabetic (DB) and non-diabetic (non-DB) patients and in cells under glucose stress, in order to elucidate whether impairment in H₂S-mediated signalling could participate in the onset of DB-related OA. Here, we identified a reduction in H₂S synthesis in the cartilage from OA-DB patients and in cells under glucose stress, which is associated with hyperglycaemia-mediated dysregulation of chondrocyte metabolism. In addition, our results indicate that H₂S is an inductor of the Nrf-2/HO-1 signalling pathway in cartilage, but is also a downstream target of Nrf-2 transcriptional activity. Thereby, impairment of the H₂S/Nrf-2 axis under glucose stress or DB triggers chondrocyte catabolic responses, favouring the disruption of cartilage homeostasis that characterizes OA pathology. Finally, our findings highlight the benefits of the use of exogeneous sources of H₂S in the treatment of DB-OA patients, and warrant future clinical studies.This research was funded by grant PI19/01206 from the Fondo de Investigación Sanitaria, integrated in the National Plan for Scientific Program, Development, and Technological Innovation 2013–2016 and funded by the Instituto de Salud Carlos III (ISCIII)-General Subdirection of Assessment and Promotion of Research-European Regional Development Fund (FEDER) “A way of making Europe”, and also by grants ED431B 2020/55 (Grupos con Potencial de Crecemento 2020) and IN607A 2021/7 (Grupos de Referencia Competitiva) from Xunta de Galicia. The study was also supported by the Biomedical Research Network Centre (CIBER), an initiative of ISCIII. C.V.-G. thanks Xunta de Galicia for his postdoctoral contract (grant number ED481D 2017/023)Xunta de Galicia; ED431B 2020/55Xunta de Galicia; IN607A 2021/7Xunta de Galicia; ED481D 2017/02

Analysis of Cryopreservation Protocols and Their Harmful Effects on the Endothelial Integrity of Human Corneas

[Abstract] Corneal cryopreservation can partially solve the worldwide concern regarding donor cornea shortage for keratoplasties. In this study, human corneas were cryopreserved using two standard cryopreservation protocols that are employed in the Tissue Bank of the Teresa Herrera Hospital (Spain) to store corneas for tectonic keratoplasties (TK protocol) and aortic valves (AV protocol), and two vitrification protocols, VS55 and DP6. Endothelial viability and general corneal state were evaluated to determine the protocol that provides the best results. The potential corneal cryopreservation protocol was studied in detail taking into consideration some cryopreservation-related variables and the endothelial integrity and stroma arrangement of the resulting cryopreserved corneas. TK corneas showed mostly viable endothelial cells, while the others showed few (AV) or none (DP6 and VS55). The corneal structure was well maintained in TK and AV corneas. TK corneas showed endothelial acellular areas surrounded by injured cells and a normal-like stromal fiber arrangement. Cryoprotectant solutions of the TK protocol presented an increasing osmolality and a physiological pH value. Cooling temperature rate of TK protocol was of 1 °C/min to −40 °C and 3 °C/min to −120 °C, and almost all of dimethyl sulfoxide left the tissue after washing. Future studies should be done changing cryopreservation-related variables of the TK protocol to store corneas of optical grade.This research was funded by Xunta de Galicia (grant no. ED431B 2020/55), by Xunta de Galicia and the European Union (European Regional Development Fund; grant nos. ED481A-2019/206 and ED481A-2017/280), and by INDITEX and the University of A Coruña (grant no. 2019)Xunta de Galicia; ED431B 2020/55Xunta de Galicia; ED481A-2019/206Xunta de Galicia; ED481A-2017/28

Cell viability assay in corneal endothelium

Resumen del póster publicado en el II Annual Meeting CINBIO abstracts book [Internet], p. 65[Abstract] Introduction: Endothelium is the inner layer of the cornea, which must be viable for transplanting. The limited availability of corneas makes necessary the developing of preservation techniques that allow a long storage without losing endothelial viability.Objectives: Optimization of a cell viability assay in preserved corneas.Methods: One half of an endothelium from a cornea that was storage in hypothermic conditions and an endothelium of a cryopreserved cornea were stained with LIVE/DEAD imaging kit and Hoechst. The other half of endothelium was the negative control. Corneal endothelia were imaged using a fluorescence microscope.Results: Four sort of cells were visualized on both endothelia: viable cells with high esterase activity, intermediate cells with low esterase activity, non-viable cells without esterase activity, and cells only stained by Hoechst. Conclusions: Triple stain is effectiveto detect different sort of cells in endothelium of preserved corneas, included viable cells, depending on their esterase enzymatic activity and on cell and nuclear membrane damage