Chondrogenic Potential of Subpopulations of Cells Expressing Mesenchymal Stem Cell Markers Derived from Human Synovial Membranes

[Abstract] In this study we analyzed the chondrogenic potential of subpopulations of mesenchymal stem cells (MSCs) derived from human synovial membranes enriched for CD73, CD106, and CD271 markers. Subpopulations of human synovial membrane MSCs enriched for CD73, CD106, and CD271 markers were isolated using a cytometry sorter and characterized by flow cytometry for MSC markers. The expression of Sox9, Nanog, and Runx2 genes by these cells was measured by reverse transcriptase-polymerase chain reaction. The chondrogenesis of each subpopulation was assessed by culturing the cells in a defined medium to produce spontaneous spheroid formation and differentiation towards chondrocyte-like cells. The examination of the spheroids by histological and immunohistochemical analyses for collagen type II (COL2), aggrecan, collagen type I (COL1), metalloprotease 13 (MMP13), and collagen type X (COLX) levels were performed to assess their chondrogenesis capacity. The adipogenesis and osteogenesis potential of each subpopulation was determined using commercial media; the resulting cells were stained with oil red O or red alizarin to test the degree of differentiation. The subpopulations had different profiles of cells positive for the MSC markers CD44, CD69, CD73, CD90, and CD105 and showed different expression levels of the genes Sox9, Nanog, and Runx2 involved in chondrogenesis, undifferentiation, and osteoblastogenesis, respectively. Immunohistochemical analysis demonstrated that COL1, COL2, COLX, MMP13, and aggrecan were expressed in the spheroids as soon as 14 days of culture. The CD271+ subpopulation expressed the highest levels of COL2 staining compared to the other subpopulations. CD105 and Runx2 were shown by immunohistochemistry and genetic analysis to have significantly higher expression CD271+ subpopulation than the other subpopulations. Spheroids formed from CD271-enriched and CD73-enriched MSCs from normal human synovial membranes mimic the native cartilage extracellular matrix more closely than CD106+ MSCs and are possible candidates for use in cartilage tissue engineering. Both cell types have potential for promoting the differentiation of MSCs into chondrocytes, presenting new possibilities for achieving intrinsic cartilage repair.Servizo Galego de Saúde; PS07/86Instituto de Salud Carlos III; CIBER BBN CB06-01-0040Instituto de Salud Carlos III; PI-08/202

Differentiation of Synovial CD-105+ Human Mesenchymal Stem Cells into Chondrocyte-like Cells through Spheroid Formation

[Abstract] Mesenchymal stem cells (MSCs) have the capacity to differentiate into several cell lineages, some of which can generate bone, cartilage, or adipose tissue. The presence of MSCs in the synovial membrane was recently reported. Data from comparative studies of MSCs derived from various mesenchymal tissues suggest that MSCs from synovial membranes have a superior chondrogenesis capacity. Previous chondrogenic differentiation studies have used the total population of MSCs, including cells with several MSC markers, such as CD44, CD90, CD105, or CD73. However the chondrogenic capacity of an individual population of MSCs has not been examined. Our aim was to study the chondrogenic capacity of the cellular MSC subset, CD105+, derived from synovial membrane tissues of patients with osteoarthritis (OA) and normal donors. The tissues were digested with a cocktail of collagenase/dispase and the isolated MSCs were seeded into plates. The subpopulation of CD105+-MSCs was separated using a magnetic separator. The MSCs were then differentiated towards chondrocyte-like cells using a specific medium to promote spheroid formation. Spheroids were collected after 14, 28, and 46 days in chondrogenic medium and stained with hematoxylin, eosin, Safranin O or Alcian blue to evaluate the extracellular matrix. Immunohistochemistry was performed to study collagen types I (COLI) and II (COLII) and aggrecan expression. Phenotypic characterization of the isolated CD105+-MSCs shows that these cells are also positive for CD90 and CD44, but negatives for CD34 and CD45. In addition, this cellular subset expressed Sox-9. Spheroids appeared after 7 days in culture in the presence of chondrogenic medium. Our studies show no differences between MSCs obtained from OA and normal synovial membranes during chondrogenesis. The morphological analysis of spheroids revealed characteristics typical of chondrocyte cells. The intensity of Safranin O, Alcian blue and aggrecan staining was positive and constant throughout the culture period. However, the intensity of COL2 staining was higher at 28 days (84.29 ± 0.1 U) than at 46 days (61.28 ± 01 U), while COL1 staining was not detected in any samples analyzed. These results were confirmed by reverse transcriptase-polymerase chain reaction assays. We conclude that the cellular subset of CD105+-MSCs has chondrogenic capacity. The study also show the similar chondrogenic capacity of CD105+-MSCs cultured from normal and OA synovial membranes. J. Cell. Biochem. 108: 145–155, 2009.Servizo Galego de Saúde; PS07/8

Umbilical Cord as a Mesenchymal Stem Cell Source for Treating Joint Pathologies

[Abstract] Articular cartilage disorders and injuries often result in life-long chronic pain and compromised quality of life. Regrettably, the regeneration of articular cartilage is a continuing challenge for biomedical research. One of the most promising therapeutic approaches is cell-based tissue engineering, which provides a healthy population of cells to the injured site but requires differentiated chondrocytes from an uninjured site. The use of healthy chondrocytes has been found to have limitations. A promising alternative cell population is mesenchymal stem cells (MSCs), known to possess excellent proliferation potential and proven capability for differentiation into chondrocytes. The “immunosuppressive” property of human MSCs makes them an important candidate for allogeneic cell therapy. The use of allogeneic MSCs to repair large defects may prove to be an alternative to current autologous and allogeneic tissue-grafting procedures. An allogeneic cell-based approach would enable MSCs to be isolated from any donor, expanded and cryopreserved in allogeneic MSC banks, providing a readily available source of progenitors for cell replacement therapy. These possibilities have spawned the current exponential growth in stem cell research in pharmaceutical and biotechnology communities. Our objective in this review is to summarize the knowledge about MSCs from umbilical cord stroma and focus mainly on their applications for joint pathologies.Ministerio de Economía y Competitividad; PLE2009-0144Instituto de Salud Carlos III; CB06-01-004

Analysis of the Chondrogenic Potential and Secretome of Mesenchymal Stem Cells Derived from Human Umbilical Cord Stroma

[Abstract] Mesenchymal stem cells (MSCs) from umbilical cord stroma were isolated by plastic adherence and characterized by flow cytometry, looking for cells positive for OCT3/4 and SSEA-4 as well as the classic MSC markers CD44, CD73, CD90, Ki67, CD105, and CD106 and negative for CD34 and CD45. Quantitative reverse transcriptase–polymerase chain reaction analysis of the genes ALP, MEF2C, MyoD, LPL, FAB4, and AMP, characteristic for the differentiated lineages, were used to evaluate early and late differentiation of 3 germ lines. Direct chondrogenic differentiation was achieved through spheroid formation by MSCs in a chondrogenic medium and the presence of chondrogenic markers at 4, 7, 14, 28, and 46 days of culture was tested. Immunohistochemistry and quantitative reverse transcriptase–polymerase chain reaction analyses were utilized to assess the expression of collagen type I, collagen type II, and collagen type X throughout the time studied. We found expression of all the markers as early as 4 days of chondrogenic differentiation culture, with their expression increasing with time, except for collagen type I, which decreased in expression in the formed spheroids after 4 days of differentiation. The signaling role of Wnt during chondrogenic differentiation was studied by western blot. We observed that β-catenin expression decreased during the chondrogenic process. Further, a secretome study to validate our model of differentiation in vitro was performed on spheroids formed during the chondrogenesis process. Our results indicate the multipotential capacity of this source of human cells; their chondrogenic capacity could be useful for future cell therapy in articular diseases.Servizo Galego de Saúde; PS07=8

A focal plane processor for continuous-time 1-D optical correlation applications

This chapter describes a 1-D Focal Plane Processor, which has been designed to run continuous-time optical correlation applications. The chip contains 200 sensory processing elements, which acquire light patterns through a 2mm ×10.9μm photodiode. The photogenerated current is scaled at the pixel level by five independent 3-bit programmable-gain current scaling blocks. The correlation patterns are defined as five sets of two hundred 3-bit numbers (from 0 to 7), which are provided to the chip through a standard I2C interface. Correlation outputs are provided in current form through 8-bit programmable gain amplifiers (PGA), whose configurations are also defined via I2C. The chip contains a mounting alignment help, which consists of three rows of 100 conventional active pixel sensors (APS) inserted at the top, middle and bottom part of the main photodiode array. The chip has been fabricated in a standard 0.35μm CMOS technology and its maximum power consumption is below 30mW. Experimental results demonstrate that the chip is able to process interference patterns moving at an equivalent frequency of 500kHz.Junta de Andalucía 2006-TIC-2352Ministerio de Ciencia e Innovación TEC2009-1181

Lamin A Deregulation in Human Mesenchymal Stem Cells Promotes an Impairment in their Chondrogenic Potential and Imbalance in their Response to Oxidative Stress

[Abstract] In the present study, we examined the effect of the over-expression of LMNA, or its mutant form progerin (PG), on the mesoderm differentiation potential of mesenchymal stem cells (MSCs) from human umbilical cord (UC) stroma using a recently described differentiation model employing spheroid formation. Accumulation of lamin A (LMNA) was previously associated with the osteoarthritis (OA) chondrocyte phenotype. Mutations of this protein are linked to laminopathies and specifically to Hutchinson–Gilford Progeria Syndrome (HGPS), an accelerated aging disease. Some authors have proposed that a deregulation of LMNA affects the differentiation potential of stem cells. The chondrogenic potential is defective in PG-MSCs, although both PG and LMNA transduced MSCs, have an increase in hypertrophy markers during chondrogenic differentiation. Furthermore, both PG and LMNA-MSCs showed a decrease in manganese superoxide dismutase (MnSODM), an increase of mitochondrial MnSODM-dependent reactive oxygen species (ROS) and alterations in their migration capacity. Finally, defects in chondrogenesis are partially reversed by periodic incubation with ROS-scavenger agent that mimics MnSODM effect. Our results indicate that over-expression of LMNA or PG by lentiviral gene delivery leads to defects in chondrogenic differentiation potential partially due to an imbalance in oxidative stress.Servizo Galego de Saúde; PS07/86Instituto de Salud Carlos III; CIBER BBNCB06-01-0040Instituto de Salud Carlos III; PI-11/0279

Implementing intelligent asset management systems (IAMS) within an industry 4.0 manufacturing environment

9th IFAC Conference on Manufacturing Modelling, Management and Control, MIM 2019; Berlin; Germany; 28 August 2019 through 30 August 2019. Publicado en IFAC-PapersOnLine 52(13), p. 2488-2493This paper aims to define the different considerations and results obtained in the implementation in an Intelligent Maintenance System of a laboratory designed based on basic concepts of Industry 4.0. The Intelligent Maintenance System uses asset monitoring techniques that allow, on-line digital modelling and automatic decision making. The three fundamental premises used for the development of the management system are the structuring of information, value identification and risk management

Biomimetic device and foreign body reaction cooperate for efficient tumour cell capture in murine advanced ovarian cancer

Metastasis is facilitated by the formation of pre-metastatic niches through the remodelling of the extracellular matrix (ECM) promoted by haematopoietic and stromal cells. The impact of these primed sites is pronounced for intraperitoneal metastases, where the cavity-exposed ECM supports the attachment of the disseminating tumour cells. Likewise, implantation of biomaterial scaffolds influences metastatic progression systemically through a foreign body reaction (FBR). In this study, we integrated the concept of creating an artificial niche to capture tumour cells actively disseminating in the peritoneal cavity with a therapeutic strategy modulating the interactions of metastatic cells with the ECM. The aim was to transform a disseminated disease into a focal disease. For this, we designed and developed a ‘biomimetic’ ECM composed of a nonresorbable three-dimensional scaffold with collagen coating and characterized the FBR to the implanted biomaterial. We also analysed the safety of the implanted devices and their ability to capture tumour cells in different murine preclinical models of advanced ovarian cancer. Implantation of the biomimetic devices resulted in an initial inflammatory reaction that transformed progressively into a fibrous connective tissue response. The adhesive capabilities of the scaffold were improved with the ancillary effect of the FBR and showed clinical utility in terms of the efficacy of capture of tumour cells, disease focalization and survival benefit. These results demonstrated the performance and safety of this ‘biomimetic’ ECM in preclinical models of advanced ovarian cancer. Translated into the clinical setting, this new therapeutic strategy represents the possibility for control of peritoneal carcinomatosis upon primary ovarian debulking surgery and to expand the percentage of patients who are candidates for second rescue surgeries at the time of relapse.This work was supported by Nasasbiotec

Proteome Analysis During Chondrocyte Differentiation in a New Chondrogenesis Model Using Human Umbilical Cord Stroma Mesenchymal Stem Cells

[Abstract] Umbilical cord stroma mesenchymal stem cells were differentiated toward chondrocyte-like cells using a new in vitro model that consists of the random formation of spheroids in a medium supplemented with fetal bovine serum on a nonadherent surface. The medium was changed after 2 days to one specific for the induction of chondrocyte differentiation. We assessed this model using reverse transcriptase-polymerase chain reaction, flow cytometry, immunohistochemistry, and secretome analyses. The purpose of this study was to determine which proteins were differentially expressed during chondrogenesis. Differential gel electrophoresis analysis was performed, followed by matrix-assisted laser desorption/ionization mass spectrometry protein identification. A total of 97 spots were modulated during the chondrogenesis process, 54 of these spots were identified as 39 different proteins and 15 were isoforms. Of the 39 different proteins identified 15 were down-regulated, 21 were up-regulated, and 3 were up- and down-regulated during the chondrogenesis process. Using Pathway Studio 7.0 software, our results showed that the major cell functions modulated during chondrogenesis were cellular differentiation, proliferation, and migration. Five proteins involved in cartilage extracellular matrix metabolism found during the differential gel electrophoresis study were confirmed using Western blot. The results indicate that our in vitro chondrogenesis model is an efficient and rapid technique for obtaining cells similar to chondrocytes that express proteins characteristic of the cartilage extracellular matrix. These chondrocyte-like cells could prove useful for future cell therapy treatment of cartilage pathologies.Ministerio de Ciencia en Innovacion; PLE2009–014

Inicio de la terapia antirretroviral en el paciente VIH a partir de un caso clínico

Nowadays HIV infection has become a potentially treatable disease, although transmission and new diagnosis rates remain high. The majority of patients reach undetectable viral replication rates and have a life expectancy similar to normal population. We present the case of a patient which was admitted to the hospital with the diagnosis of pneumonia caused by Pneumocystis jirovecii and was later diagnosed with HIV infection. According to the latest evidence, early initiation of antiretroviral therapy is the most beneficial and recommended management for patients with HIV diagnosis, not only because of benefits for the patient, but to reduce transmission.En la actualidad, la infección por el virus de la inmunodeficiencia humana (VIH) se considera una enfermedad crónica tratable, en la que se ha conseguido que la mayoría de los pacientes alcancen la supresión virológica y tengan una esperanza de vida equiparable a la de la población general. No obstante, casi la mitad de los nuevos diagnósticos siguen siendo en personas con enfermedad avanzada. Presentamos el caso de un paciente que debutó con una neumonía por Pneumocystis jirovecii y fue diagnosticado de infección por VIH. Según las últimas evidencias, el inicio de la terapia antirretroviral debe ser lo más precoz posible, tanto por los beneficios sobre el paciente como para evitar la transmisión de la infección.