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

First record of Clavulina fuscolilacina (Cantharellales, Basidiomycota) for Mexico

Antecedentes y Objetivos: El género Clavulina pertenece a la familia Hydnaceae (Cantharellales, Basidiomycota). Estos hongos presentan hábitos ectomicorrizógenos y se distribuyen tanto en bosques templados como tropicales, siendo estos últimos donde se encuentra la mayor diversidad de especies. El presente documento tiene como finalidad contribuir al conocimiento de la funga tropical mexicana, en específico de la Península de Yucatán. Métodos: La recolección de los ejemplares se llevó a cabo en los estados de Quintana Roo y Yucatán en vegetación costera y selva baja caducifolia, respectivamente. Se siguieron los protocolos micológicos generales para la recolección, caracterización y curación de los basidiomas. El material revisado se encuentra depositado en los herbarios ITCV, del Instituto Tecnológico de Ciudad Victoria y UADY, de la Universidad Autónoma de Yucatán. Resultados clave: Clavulina fuscolilacina se caracteriza por sus basidiomas claviformes o ramificados, de color lila o gris, que crecen gregarios bajo Polygonaceae como Coccoloba uvifera y Gymnopodium floribundum, posiblemente en asociación ectomicorrizógena. Previamente solo se conocía de Brasil; con este trabajo se amplía su rango de distribución hasta México. Además, se presenta una clave taxonómica para el género Clavulina en México. Conclusiones: Este es el primer registro del género Clavulina para la Península de Yucatán. Por otro lado, es la primera vez que se cita C. fuscolilacina para México y fuera de Brasil.Background and Aims: The genus Clavulina belongs to the Hydnaceae family (Cantharellales, Basidiomycota). They have ectomycorrhizal habits and are located in both temperate and tropical forests; the highest species diversity is found in the latter. The purpose of this document is to contribute to the knowledge of the Mexican tropical funga, specifically in the Yucatán Península. Methods: Collection of the specimens was carried out in the states of Quintana Roo and Yucatán, in coastal vegetation and deciduous lowland tropical forests, respectively. The general mycological protocols for collecting, characterizing, and curating the basidiomas were followed. The specimens are deposited in the herbaria ITCV of the Instituto Tecnológico de Ciudad Victoria and UADY of the Universidad Autónoma de Yucatán. Key results: Clavulina fuscolilacina is characterized by claviform or branched basidiomas, lilac or gray in color. It grows gregarious under Polygonaceae like Coccoloba uvifera and Gymnopodium floribundum, possibly forming an ectomycorrhizal association. Previously, this species was known only from Brazil, so this record expands its distribution range to Mexico. Moreover, a taxonomic key of the genus Clavulina for Mexico is presented. Conclusions: The first record of the genus Clavulina for the Yucatán Península is presented. Furthermore, this is the first report of C. fuscolilacina from Mexico and beyond Brazil

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

Two new species of Trichoglossum (Geoglossaceae, Ascomycota) from south Mexico

Two new species of Trichoglossum are described from south Mexico based on morphological and molecular evidence. Trichoglossum caespitosum is characterized by the caespitose ascomata, rough and coiled paraphy-ses and the ascospores with 9-11 septa. Trichoglossum tropicale is characterized by the capitate ascomata, clavate and straight paraphyses and the ascospores with 10-12 septa. Both species grow in the tropical forests of the Yucatan peninsula. Here we provide descriptions and photographs for these species, together with a phylogenetic analyses based on the DNA sequences of nuc rDNA (ITS region and 28S gene) and a comparative table for the species known for America

Melanogaster coccolobae sp. nov. (Paxillaceae, Boletales), un hongo hipogeo tropical de las áreas urbanas de Quintana Roo, México

Background and Aims: The genus Melanogaster is characterized by its hypogeous to semi hypogeous habit, brownish basidiomata, gel-filled gleba locules, and globose to ellipsoid basidiospores. The genus is distributed in temperate zones, but sequences from Coccoloba root tips and a few basidiome collections have revealed its presence in the tropics. The aim of this article is to describe a new species of Melanogaster based on ecological, molecular, and morphological data. Methods: Specimens were collected in urban vegetation of Quintana Roo in the Yucatán Peninsula, Mexico. For morphological description, the classic protocols for sequestrate fungi were followed. The dried material was deposited in the mycological herbarium “José Castillo Tovar” of the Instituto Tecnológico de Ciudad Victoria (ITCV) and the herbarium of the Universidad Autónoma de Yucatán (UADY). Key results: Melanogaster coccolobae is presented as a new species from the urban gardens of Quintana Roo based on ecological, molecular, and morphological evidence. This species is characterized by its hypogeous to semi hypogeous basidioma, greyish orange, brown to reddish brown peridium composed of two layers, sweet smell, subglobose, ellipsoid or piriform basidiospores, and by its mycorrhizal association with Coccoloba spicata. Conclusions: Melanogaster coccolobae is the first species described from the Mexican Caribbean from urban gardens with Coccoloba spicata. More studies about the tropical sequestrate fungi are recommended.Antecedentes y Objetivos: El género Melanogaster se caracteriza por su hábito hipogeo a semi hipogeo, basidiomas parduscos, gleba con lóculos llenos de gel y basidiosporas globosas a elipsoides. El género se distribuye en zonas templadas, pero secuencias de ectomicorrizas de Coccoloba y pocas colecciones de basidiomas han revelado su presencia en los trópicos. El objetivo de este artículo es describir una nueva especie de Melanogaster a partir de datos ecológicos, moleculares y morfológicos. Métodos: Los especímenes fueron recolectados en jardines urbanos de Quintana Roo en la Península de Yucatán, México. Para la descripción morfológica se siguieron los protocolos clásicos para hongos secuestrados. El material se depositó en el herbario micológico “José Castillo Tovar” del Instituto Tecnológico de Ciudad Victoria (ITCV) y en el herbario de la Universidad Autónoma de Yucatán (UADY). Resultados clave: Melanogaster coccolobae se presenta como una nueva especie de los jardines urbanos de Quintana Roo con base en evidencia morfológica, ecológica y molecular. Esta especie se caracteriza por sus basidiomas hipogeos a semi hipogeos, peridio naranja grisáceo, marrón o marrón rojizo, compuesto por dos capas, olor dulce, basidiosporas subglobosas, elipsoides o piriformes y por formar asociación micorrízica con Coccoloba spicata. Conclusiones: Melanogaster coccolobae es la primera especie descrita del Caribe mexicano en jardines urbanos con Coccoloba spicata. Se recomiendan más estudios sobre los hongos secuestrados tropicales

Pulmonary vascular remodeling and prognosis in patients evaluated for heart transplantation: insights from the OCTOPUS-CHF study

[Abstract] Objective: In patients with advanced heart failure, the intravascular optical coherence tomography (OCT) of subsegmental pulmonary artery measurements is correlated with right heart catheterization parameters. Our aim was to study the prognostic value of pulmonary OCT, right heart catheterization data, and the echocardiographic estimation of pulmonary pressure in patients studied for elective heart transplants. Methods: This research is an observational, prospective, multicenter study involving 90 adults with a one-year follow-up. Results: A total of 10 patients (11.1%) died due to worsening heart failure before heart transplantation, 50 underwent a heart transplant (55.6%), and 9 died in the first year after the transplant. The patients with and without events (mortality or heart failure-induced hospitalization) had similar data regarding echocardiography, right heart catheterization, and pulmonary OCT (with a median estimated pulmonary artery systolic pressure of 42.0 mmHg, interquartile range (IQR) of 30.3-50.0 vs. 47.0 mmHg, IQR 34.6-59.5 and p = 0.79, median pulmonary vascular resistance of 2.2 Wood units, IQR 1.3-3.7 vs. 2.0 Wood units, IQR 1.4-3.2 and p = 0.99, and a median pulmonary artery wall thickness of 0.2 ± 0.5 mm vs. 0.2 ± 0.6 mm and p = 0.87). Conclusion: Pulmonary vascular remodeling (evaluated with echocardiography, right heart catheterization, and pulmonary OCT) was not associated with prognosis in a selected sample of adults evaluated for elective heart transplants. Pulmonary OCT is safe and feasible for the evaluation of these patients.Instituto de Salud Carlos III; PI18/00254European Regional Development Fund; CB16/11/0050

Pulmonary Vascular Remodeling and Prognosis in Patients Evaluated for Heart Transplantation: Insights from the OCTOPUS-CHF Study

Objective: In patients with advanced heart failure, the intravascular optical coherence tomography (OCT) of subsegmental pulmonary artery measurements is correlated with right heart catheterization parameters. Our aim was to study the prognostic value of pulmonary OCT, right heart catheterization data, and the echocardiographic estimation of pulmonary pressure in patients studied for elective heart transplants. Methods: This research is an observational, prospective, multicenter study involving 90 adults with a one-year follow-up. Results: A total of 10 patients (11.1%) died due to worsening heart failure before heart transplantation, 50 underwent a heart transplant (55.6%), and 9 died in the first year after the transplant. The patients with and without events (mortality or heart failure-induced hospitalization) had similar data regarding echocardiography, right heart catheterization, and pulmonary OCT (with a median estimated pulmonary artery systolic pressure of 42.0 mmHg, interquartile range (IQR) of 30.3-50.0 vs. 47.0 mmHg, IQR 34.6-59.5 and p = 0.79, median pulmonary vascular resistance of 2.2 Wood units, IQR 1.3-3.7 vs. 2.0 Wood units, IQR 1.4-3.2 and p = 0.99, and a median pulmonary artery wall thickness of 0.2 +/- 0.5 mm vs. 0.2 +/- 0.6 mm and p = 0.87). Conclusion: Pulmonary vascular remodeling (evaluated with echocardiography, right heart catheterization, and pulmonary OCT) was not associated with prognosis in a selected sample of adults evaluated for elective heart transplants. Pulmonary OCT is safe and feasible for the evaluation of these patients