Ingeniería Tisular del hueso y el cartílago empleando células mesenquimales estromales y biomateriales

Programa Oficial de Doutoramento en Ciencias da Saúde. 5007V01[Resumo]A artrose (OA) é a patoloxía reumática máis prevalente e, na actualidade, non existe un tratamento efectivo. Unha das causas deste trastorno é o desenvolvemento secundario tras unha lesión na cartilaxe hialina articular ou no óso subcondral. A Enxeñería Tisular (IT) osteocondral, utilizando células e soportes, xurdiu como unha terapia prometedora. O obxectivo deste estudo foi determinar o potencial de reparación das células mesenquimais estromais (CMEs), en combinación con diferentes tipos de soportes, para o seu uso na IT osteocondral. En primeiro lugar, illáronse e caracterizáronse CMEs, obtidas de medula ósea humana e ovina, e de células similares a CMEs (CSMEs), diferenciadas a partir de células pluripotentes inducidas (iPS). En segundo lugar, estudouse a capacidade de diferenciación condroxénica das CMEs humanas, cultivadas en diferentes soportes de coláxeno (Col). Tamén, testouse a capacidade de diferenciación osteoxénica das CMEs ovinas, cultivadas sobre soportes de Col I e β fosfato tricálcico (β-FTC). Por último, analizouse a capacidade de reparación das CMEs, cultivadas en soportes de Col, nun modelo de lesión de cartilaxe in vitro. Os resultados confirman a identidade das CMEs e a súa capacidade de formar neotecidos condroxénicos e osteoxénicos sobre biomateriais de Col. A presenza de proteoglicans (PGs) na composición dos soportes mellora o fenotipo condroxénico do tecido cartilaxinoso neoformado e a reparación de lesións na cartilaxe hialina, nun modelo in vitro. O uso dos constructos, formados polas CMEs e biomateriais de Col, resulta prometedor para a reparación de lesións osteocondrais.[Resumen]La artrosis (OA) es la patología reumática más prevalente y, actualmente, no existe un tratamiento efectivo. Una de las causas de la enfermedad es su desarrollo secundario tras una lesión en el cartílago hialino articular o en el hueso subcondral. La Ingeniería Tisular (IT) osteocondral, utilizando células y soportes, ha surgido como una terapia prometedora. El objetivo de este estudio fue determinar el potencial de reparación de las células mesenquimales estromales (CMEs), en combinación con diferentes tipos de soportes, para su uso en la IT osteocondral. En primer lugar, se aislaron y caracterizaron CMEs, obtenidas de médula ósea humana y ovina, y de células similares a CMEs (CSMEs), diferenciadas a partir de células pluripotentes inducidas (iPS). En segundo lugar, se estudió la capacidad de diferenciación condrogénica de las CMEs humanas, cultivadas en diferentes soportes de colágeno (Col). También, se testó la capacidad de diferenciación osteogénica de las CMEs ovinas, cultivadas sobre soportes de Col I y β fosfato tricálcico (β-FTC). Por último, se analizó la capacidad de reparación de las CMEs, cultivadas en soportes de Col, en un modelo de lesión de cartílago in vitro. Los resultados confirman la identidad de las CMEs y su capacidad de formar neotejidos condrogénicos y osteogénicos sobre biomateriales de Col. La presencia de proteoglicanos (PGs) en la composición de los soportes mejora el fenotipo del tejido cartilaginoso neoformado y la reparación de lesiones en el cartílago hialino, en un modelo in vitro. El uso de los constructos, formados por las CMEs y biomateriales de Col, resulta prometedor para la reparación de lesiones osteocondrales.[Abstract]Osteoarthritis (OA) is the most prevalent rheumatic disorder and currently, there is no effective treatment to treat it. Lesions in the articular hyaline cartilage or in the subchondral bone may lead secondarily to OA. Osteochondral Tissue Engineering (IT) using cells and scaffolds has emerged as a promising therapy. The aim of this study was to determine the repair potential of mesenchymal stromal cells (CMEs) combined with different types of scaffolds, and their usefulness in osteochondral IT. First, CMEs from human and ovine bone marrow and, from induced plutipotent stem cells (iPS) were isolated and characterized. Secondly, chondrogenic differentiation capacity of human CMEs on collagen (Col) scaffolds was studied. Also, osteogenic differentiation capacity of ovine CMEs on Col I and β tricalcium phosphate (β-FTC) was tested. Finally, the repair capacity of CMEs cultured on Col scaffolds in an in vitro cartilage lesion model was assessed. Results confirmed the CMEs identity and their capacity to form chondrogenic and osteogenic neotissues, when cultured on Col scaffolds. The addition of proteoglycans (PGs) to the scaffold composition improves the phenotype of neoformed cartilaginous tissue and the repair capacity in the in vitro hyaline cartilage lesion model. The use of CMEs and Col scaffolds for engineered tissue constructs is a promising approach for osteochondral lesions repair

Diferenciación "in vitro" de células madre aisladas de médula ósea humana sobre biomateriales de colágeno

Objetivos. El objetivo de este estudio ha sido evaluar la idoneidad de diferentes biomateriales de colágeno como posibles soportes de ingeniería tisular, para el tratamiento de lesiones cartilaginosas. Metodología. Se aislaron células madre mesenquimales de la médula ósea humana (CMMs-MOh), de varios donantes, y se expandieron in vitro. Tras obtener un número suficiente de células, se formó un conjunto celular y se sembró sobre los biomateriales, con un medio de estimulación de la diferenciación condrogénica. Tras 30 días de estimulación se testó el crecimiento y diferenciación mediante microscopía electrónica, técnicas histológicas, técnicas inmunohistoquímicas y biología celular. Resultados. Todos los biomateriales presentaron crecimiento y proliferación celular. En todos los biomateriales tuvo lugar diferenciación condrogénica de las CMM-MOh, en mayor o menor medida. Conclusiones. Todos los biomateriales permitieron el crecimiento y proliferación de las CMMs-MOh, pero sólo en el biomaterial C1C2HS se observó formación de tejido con características similares a cartílago. Futuros estudios permitirán determinar si éste biomaterial es adecuado para la reparación de cartílago articular.Traballo fin de mestrado (UDC.FCS). Asistencia e investigación sanitaria. Especialidade en fundamentos de investigación biomédica. Curso 2011/201

Chondrogenic differentiation of human mesenchymal stem cells via SOX9 delivery in cationic niosomes

[Abstract] Gene transfer to mesenchymal stem cells constitutes a powerful approach to promote their differentiation into the appropriate cartilage phenotype. Although viral vectors represent gold standard vehicles, because of their high efficiency, their use is precluded by important concerns including an elevated immunogenicity and the possibility of insertional mutagenesis. Therefore, the development of new and efficient non-viral vectors is under active investigation. In the present study, we developed new non-viral carriers based on niosomes to promote the effective chondrogenesis of human MSCs. Two different niosome formulations were prepared by varying their composition on non-ionic surfactant, polysorbate 80 solely (P80), or combined with poloxamer 407 (P80PX). The best niosome formulation was proven to transfer a plasmid, encoding for the potent chondrogenic transcription factor SOX9 in hMSC aggregate cultures. Transfection of hMSC aggregates via nioplexes resulted in an increased chondrogenic differentiation with reduced hypertrophy. These results highlight the potential of niosome formulations for gene therapy approaches focused on cartilage repair.Ministerio de Ciencia e Innovación (España); RTI2018-099389-A-100Ministerio de Ciencia e Innovación (España); RYC2018-025617-IXunta de Galicia; ED431F2021/1

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

Generation of Mesenchymal Cell Lines Derived from Aged Donors

[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; R2016/036Xunta de Galicia; ED431B 2020/5

Tips and Tricks for Successfully Culturing and Adapting Human Induced Pluripotent Stem Cells [Protocol]

Protocol[Abstract] 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.We thank the laboratory staff from INIBIC-CHUAC, the Radio Physics department from the Oncology Center of Galicia and the Servicio de Xenética (CHUAC) for their assistance. We also thank the staff from Oza University Library (University of A Coruña) for their collaboration. We also thank our funding agencies: Instituto de Salud Carlos III-General Subdirection of Assessment and Promotion of the Research—European Regional Development Fund (FEDER) “A way of making Europe” (PI17/02197 and PI20/00933); Rede Galega deTerapia Celular and Grupos con Potencial de Crecemento, Xunta de Galicia (R2016/036, R2014/050, CN2012/142, ED431B 2020/55, and GPC2014/048); the University of A Coruña; M.P.-R. and S.R.-F. are granted by a predoctoral fellowship from Xunta de Galicia and European Union (European Social Fund) and C.S.-R. was beneficiary of a postdoctoral fellowship from Xunta de GaliciaXunta de Galicia; R2016/036Xunta de Galicia; R2014/050Xunta de Galicia; CN2012/142Xunta de Galicia; ED431B 2020/55Xunta de Galicia; GPC2014/04

Immortalizing mesenchymal stromal cells from aged donors while keeping their essential features

[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; R2016/036Xunta de Galicia; R2014/050Xunta de Galicia; CN2012/142Xunta de Galicia; GPC2014/048Deputación da Coruña; BINV-CS/2016Instituto de Salud Carlos III; PI17/0219

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