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.

Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations

“…In both subpopulations, an MSC profile was consistent [26]. Both subpopulations were able to initiate differentiation.…”

“…The amniotic membrane was mechanically separated from the underlying chorion by blunt dissection. Membrane fragments were subjected to enzymatic digestion [26]. For cell sorting, a single suspension of AM-hMSCs (7 x10 6 ) was stained with anti-human specific antibodies CD73-phycoerythrin (PE) (BioLegend; San Diego, CA, USA), CD44-biotin (Serotec; Kidlington, UK) and CD105-APC (BioLegend; San Diego, CA, USA).…”

“…In our previous study, we isolated the CD105 + and CD105 -subpopulations, characterized their immunophenotype, and demonstrated their osteoblastic differentiation ability [26]. To describe the temporality of each of the three stages of osteoblastic differentiation induced by dexamethasone, we analyzed molecular and cellular markers in both subpopulations.…”

“…Both subpopulations were able to initiate differentiation. However, differences in the time required to reach osteoblastic maturity and mineralization efficiency during induction with dexamethasone were evidenced [26].…”

Effect on growth and osteoblast mineralization of hydroxyapatite-zirconia (HA-ZrO 2 ) obtained by a new low temperature system

“…Isolation of hMSCs and osteoblast cell culture hMSCs were isolated as described by Leyva et al [26]. Cell seeding on scaffolds Microspheres of HA-ZrO 2 -alginate were prepared in two steps: 0.075 g of natural bone matrix fine powder (NBM) and 0.15 g of HA-ZrO 2 (HZ600, HZ650 or HZ700) were mixed with the osteoblast (1×10 4 cells) resuspended in a solution of 1.2% alginate/DMEM.…”