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Leyva-Leyva M.,Laboratorio Of Biologia Molecular E Histocompatibilidad | Barrera L.,Instituto Nacional Of Enfermedades Respiratorias Ismael Cosio Villegas | Lopez-Camarillo C.,National Autonomous University of Mexico | Arriaga-Pizano L.,Centro Medico Nacional | And 9 more authors.
Stem Cells and Development | Year: 2013

Human fetal mesenchymal stem cells can be isolated from the amniotic membrane (AM-hMSCs) by enzymatic digestion. The biological properties of this cell population have been characterized; however, few studies have focused on the presence of stem cell subpopulations and their differentiation potential. The aim of the present study was to isolate homogeneous AM-hMSC subpopulations based on the coexpression of surface markers. In addition, we aimed to characterize stem cell subpopulations through the detection of typical stem cell markers and its differentiation potential. In this study, fluorescence- activated cell sorting (FACS) was used to positively select for the surface markers CD44, CD73, and CD105. Two subpopulations were isolated: CD44 +/CD73+/CD105+ (CD105+), and CD44+/CD73+/CD105- (CD105-). To characterize the cell subpopulations, the expression of pluripotency-associated markers was analyzed by reverse transcriptase-polymerase chain reaction and immunofluorescence. Our results showed positive expression of SOX2, SOX3, PAX6, OCT3/4, and NANOG in the CD105+ and CD105- cell subpopulations. In contrast, we did not detect expression of SSEA4 or FOXD3 in either subpopulation. Immunophenotypes, such as mesenchymal and hematopoietic markers, were studied by FACS analyses. Our data revealed the expression of the CD49a, CD49d, CD29, integrin α9β1, CD44, CD73, and CD105 antigens in both subpopulations. In contrast, CD90, CD45, CD34, CD14, and HLA-DR expression was not detected. The ability of both subpopulations to differentiate into osteoblasts, adipocytes, and chondrocytes was evidenced using Alizarin red, Oil-Red, and Alcian blue staining, respectively. Furthermore, neuronal differentiation was demonstrated by the expression of GFAP and NEURO-D. Interestingly, we observed a dissimilar osteoblastic differentiation potential between the subpopulations. CD105- cells showed stronger expression of secreted protein acidic and rich in cysteine (SPARC) and osteonectin, which was associated with more effective calcium deposition, than CD105+ cells. In conclusion, we described a systematic method for the isolation of hMSCs that was highly reproducible and generated homogeneous cultures for osteoblast differentiation with an efficient capacity for mineralization. © Mary Ann Liebert, Inc.

Leyva-Leyva M.,Laboratorio Of Biologia Molecular E Histocompatibilidad | Lopez-Diaz A.,Laboratorio Of Biologia Molecular E Histocompatibilidad | Barrera L.,Instituto Nacional Of Enfermedades Respiratorias Ismael Cosio Villegas | Camacho-Morales A.,Autonomous University of Nuevo Leon | And 17 more authors.
Stem Cells and Development | Year: 2015

Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105+ and CD105-) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105- cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105+ cells at the maturation stage. Interestingly, levels of activated β1-integrin increased in CD105- cells during the process. Additionally, the CD105- subpopulation showed 3-fold increase of phosphorylated FAKY397 compared to CD105+ cells. Results also indicate that ERK1/2 was activated during CD105- bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105+ differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105- showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105+ showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,397 and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105- subpopulation showed superior adhesion efficiency compared to CD105+ cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105- subpopulation at the maturation stage represents an attractive strategy for clinical use in orthopedic bioengineering. © 2015 Mary Ann Liebert, Inc.

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