title: Characterization of mesenchymal stem cell subpopulations from human amniotic membrane with dissimilar osteoblastic potential.
abstract: 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.
authors.affiliation: Laboratorio de Biología Molecular e Histocompatibilidad, Dirección de Investigación Hospital General Dr. Manuel Gea González, México, México.
authors.forename: Margarita
authors.initials: M
authors.lastname: Leyva-Leyva
authors.forename: Lourdes
authors.initials: L
authors.lastname: Barrera
authors.forename: César
authors.initials: C
authors.lastname: López-Camarillo
authors.forename: Lourdes
authors.initials: L
authors.lastname: Arriaga-Pizano
authors.forename: Gabriel
authors.initials: G
authors.lastname: Orozco-Hoyuela
authors.forename: Erika M
authors.initials: EM
authors.lastname: Carrillo-Casas
authors.forename: Jaime
authors.initials: J
authors.lastname: Calderón-Pérez
authors.forename: Annia
authors.initials: A
authors.lastname: López-Díaz
authors.forename: Felipe
authors.initials: F
authors.lastname: Hernandez-Aguilar
authors.forename: Ricardo
authors.initials: R
authors.lastname: González-Ramírez
authors.forename: Simón
authors.initials: S
authors.lastname: Kawa
authors.forename: Jesús
authors.initials: J
authors.lastname: Chimal-Monroy
authors.forename: Lizeth
authors.initials: L
authors.lastname: Fuentes-Mera
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism; D000650:Amnion / Q000166:cytology* / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation; D002469:Cell Separation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D000071063:Endoglin; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D018613:Microscopy, Confocal; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D015676:Osteonectin / Q000235:genetics / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism; D011247:Pregnancy; D011956:Receptors, Cell Surface / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; C064206:ENG protein, human; D000071063:Endoglin; D005136:Eye Proteins; D018398:Homeodomain Proteins; D018960:Hyaluronan Receptors; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D015676:Osteonectin; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; C494173:POU5F1 protein, human; D051761:Paired Box Transcription Factors; D011956:Receptors, Cell Surface; D012097:Repressor Proteins; C527782:SOX2 protein, human; C527785:SOX3 protein, human; D055748:SOXB1 Transcription Factors; C577241:SPARC protein, human; D015675:Osteocalcin; D015720:5'-Nucleotidase