Multilineage differentiation potential of cells isolated from the human amniotic membrane

Abstract

The human amniotic membrane (HAM) contains two cell types from different embryological origins. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. In this study, we localized, isolated, quantified and phenotypically characterized HAM-derived cells and analysed their in vitro differentiation potential towards mesodermal cell lineages. Human amnion-derived cells were isolated and characterized by flow cytometry. Immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction studies were performed for the analysis of multipotentiality. Immunophenotypic characterization of both cell types demonstrated the presence of the common, well-defined human mesenchymal stem cell (MSC) markers (CD90, CD44, CD73, CD166, CD105, CD29), as well as the embryonic stem-cell markers SSEA-4 and STRO-1. Phenotypes of both cell populations were maintained from passages P0 to P9. The assessment of multilineage potential demonstrated that the hAMSCs showed greater adipogenic and chondrogenic potential. Both populations had the ability to retain their capacity for differentiation during culture passages from P0 to P4. Our data demonstrate the successful localization and isolation of hAMSCs and hAECs from the HAM. Both cell populations possessed similar immunophenotype. However, they differed in cell yield and multipotential for differentiation into the major mesodermal lineages. Our functional differentiation studies demonstrated that hAMSCs possess a much greater mesodermal differentiation capacity than hAECs. These considerations will be important for use of these cells for cell therapy.

Exploring the Human Term Placenta as a Novel Source for Stem Cells and their Application in the Clinic

“…Thus, they may be a superior MSC source for clinical trials than the traditional source which is adult bone marrow. This section will focus on the isolation of MSC from amniotic fluid (Mareschi et al, 2009, Nadri and Soleimani, 2007, Roubelakis et al, 2007, the amniotic membrane (Soncini et al, 2007, Portmann-Lanz et al, 2006, Diaz-Prado et al, 2010, the chorion (chorion laeve and chorionic villi) (Soncini et al, 2007, Portmann-Lanz et al, 2006, Igura et al, 2004, Poloni et al, 2008, the decidua (Macias et al, 2010, Aghajanova et al, 2010, umbilical cord/Wharton's jelly (Mitchell et al, 2003, Reinisch et al, 2007, Petsa et al, 2009) and umbilical cord blood (Mareschi et al, 2001, Bieback et al, 2004, Romanov et al, 2003. This section will also discuss the isolation of haematopoietic stem cells from the umbilical cord blood and the application of these cells.…”

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“…Antibody permeability was increased by incubation with a detergent (0.1% Triton X-100, Sigma-Aldrich, St. Louis, MO, USA) in phosphatebuffered saline (30 min Isolation and culture of human amniotic mesenchymal stromal cells Human amniotic mesenchymal stromal cells were isolated and cultured with a modification of a previously described method. [17][18] The amnion was peeled away and processed separately from the chorion and washed in phosphate-buffered saline with 1% penicillin and streptomycin (Gibco BRL, Life Technologies, Grand Island, NY, USA) to remove red blood cells. For isolation of human amniotic mesenchymal stromal cells, amnions were dissected, fragmented (2-3 mm 3 pieces), and incubated (15 min, 37ºC) in Dulbecco Modified Eagle Medium with low glucose (Gibco BRL, Life Technologies) and 0.25% trypsin (Gibco BRL, Life Technologies) to release amniotic epithelial cells.…”

Differentiation of hESCs into Mesodermal Subtypes: Vascular-, Hematopoietic- and Mesenchymal-lineage Cells

“…Since MSCs were first derived from bone marrow in the adult human body (46), MSCs with similar characteristics have been collected from various other tissues, including peripheral blood and adipose tissue (47,48). Recently, MSCs have also been derived from fetal sources, such as cord blood and amnion (49,50). In general, MSCs are characterized by the combinatorial expression patterns of various surface markers, including CD105, CD73, CD166, HLA Class I, CD44, CD 146 and CD90, which are positively expressed.…”

“…Conversely, CD45, CD34, CD14, CD31, CD19 and HLA-DR (51, 52) are not expressed by MSCs. Although MSCs do not have defined unique characteristics, unlike more common specific cell types, because of their high proliferation and multipotent differentiation capacity, MSCs are now being used in many areas as a cell source for regenerative therapy (47,49,50). However, adult MSCs display irregular proliferation patterns when sub-cultured for long periods, thus requiring repeated preparations from adults, making research on the topic more difficult (53).…”