In vitro and in vivo properties of distinct populations of amniotic fluid mesenchymal progenitor cells

Cell and Gene Therapy Laboratory, Centre of Basic Research, Biomedical Research Foundation, Academy of Athens (BRFAA), Athens, Greece.
Journal of Cellular and Molecular Medicine (Impact Factor: 3.7). 09/2010; 15(9):1896-913. DOI: 10.1111/j.1582-4934.2010.01180.x
Source: PubMed

ABSTRACT Human mesenchymal progenitor cells (MPCs) are considered to be of great promise for use in tissue repair and regenerative medicine. MPCs represent multipotent adherent cells, able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes or chondrocytes. Recently, we identified and characterized human second trimester amniotic fluid (AF) as a novel source of MPCs. Herein, we found that early colonies of AF-MPCs consisted of two morphologically distinct adherent cell types, termed as spindle-shaped (SS) and round-shaped (RS). A detailed analysis of these two populations showed that SS-AF-MPCs expressed CD90 antigen in a higher level and exhibited a greater proliferation and differentiation potential. To characterize better the molecular identity of these two populations, we have generated a comparative proteomic map of SS-AF-MPCs and RS-AF-MPCs, identifying 25 differentially expressed proteins and 10 proteins uniquely expressed in RS-AF-MPCs. Furthermore, SS-AF-MPCs exhibited significantly higher migration ability on extracellular matrices, such as fibronectin and laminin in vitro, compared to RS-AF-MPCs and thus we further evaluated SS-AF-MPCs for potential use as therapeutic tools in vivo. Therefore, we tested whether GFP-lentiviral transduced SS-AF-MPCs retained their stem cell identity, proliferation and differentiation potential. GFP-SS-AF-MPCs were then successfully delivered into immunosuppressed mice, distributed in different tissues and survived longterm in vivo. In summary, these results demonstrated that AF-MPCs consisted of at least two different MPC populations. In addition, SS-AF-MPCs, isolated based on their colony morphology and CD90 expression, represented the only MPC population that can be expanded easily in culture and used as an efficient tool for future in vivo therapeutic applications.

Download full-text


Available from: Maria G Roubelakis, Oct 21, 2014
  • Source
    • "First, we isolated clones of long-lived epithelial cells; PB3B5 cells have been in culture for more than 25 passages. This is significant because others have noted that clonal populations of epithelial cells are difficult to maintain beyond 5 or 6 passages [35] [40] [41] and that amniotic cell cultures either show or acquire a uniform stromal or fibroblast-like morphology during culture [26, 42–44]. Long-lived clonal populations like PB3B5 could reflect unique epithelial cell types and/or propagation of epithelial cell clones without the paracrine effects that may be present in mixed cell populations. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fetal cells are widely considered a superior cell source for regenerative medicine; fetal cells show higher proliferative capacity and have undergone fewer replicative cycles that could generate spontaneous mutations. Fetal cells in amniotic fluid were among the first normal primary cells to be cultured ex vivo, but the undefined composition of amniotic fluid has hindered advance for regenerative applications. We first developed a highly efficient method to generate clonal populations by dilution of amniocentesis samples in media and direct plating without intervening refrigeration, centrifugation, or exposure of cells to the paracrine effects in mixed cell cultures. More than 40 clonal populations were recovered from 4 amniocentesis samples and representative clones were characterized by flow cytometry, conventional assays for differentiation potential, immunofluorescence imaging, and transcript analysis. The results revealed previously unreported diversity among stromal and epithelial cell types and identified unique cell types that could be lost or undetected in mixed cell populations. The differentiation potential of amniotic cells proved to be uncoupled from expression of definitive cell surface or cytoplasmic markers for stromal and epithelial cells. Evidence for diversity among stromal and epithelial cells in amniotic fluid bears on interpretations applied to molecular and functional tests of amniotic cell populations.
    09/2012; 2012(4):485950. DOI:10.1155/2012/485950
  • Source
    • "Up to date, a number of different cultivation protocols have been reported, leading to enriched stem cell populations. The isolation of AFSC and the respective culture protocols were summarized in a recent review by Klemmt et al. [3] and can be categorized as follows: (i) a single step cultivation protocol, where the primary culture was left undisturbed for 7 days or more until the first colonies appear [2, 3, 30–32], (ii) a two-step cultivation protocol, where amniocytes, not attached after 5 days in culture, were collected and further expanded [3] [5] [33], (iii) cell surface marker selection for CD117 (c-kit receptor) [3] [7] [34] [35], (iv) mechanical isolation of the initial mesenchymal progenitor cell colonies formed in the initial cultures [9], and (v) short-term cultures to isolate fibroblastoid colonies [36]. The majority of the AFSCs, isolated following these methodologies, shared a multipotent mesenchymal phenotype and exhibited higher proliferation potential and a wider differentiation potential compared to adult MSCs [2, 4–7, 9, 24, 37]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Amniotic fluid (AF) and amniotic membrane (AM) have been recently characterized as promising sources of stem or progenitor cells. Both not only contain subpopulations with stem cell characteristics resembling to adult stem cells, such as mesenchymal stem cells, but also exhibit some embryonic stem cell properties like (i) expression of pluripotency markers, (ii) high expansion in vitro, or (iii) multilineage differentiation capacity. Recent efforts have been focused on the isolation and the detailed characterization of these stem cell types. However, variations in their phenotype, their heterogeneity described by different groups, and the absence of a single marker expressed only in these cells may prevent the isolation of a pure homogeneous stem cell population from these sources and their potential use of these cells in therapeutic applications. In this paper, we aim to summarize the recent progress in marker discovery for stem cells derived from fetal sources such as AF and AM, using novel methodologies based on transcriptomics, proteomics, or secretome analyses.
    06/2012; 2012:107836. DOI:10.1155/2012/107836
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Amniotic fluid (AF) has become an interesting source of fetal stem cells. However, AF contains heterogeneous and multiple, partially differentiated cell types. After isolation from the amniotic fluid, cells were characterized regarding their morphology and growth dynamics. They were sorted by magnetic associated cell sorting using the surface marker CD 117. In order to show stem cell characteristics such as pluripotency and to evaluate a possible therapeutic application of these cells, AF fluid-derived stem cells were differentiated along the adipogenic, osteogenic, and chondrogenic as well as the neuronal lineage under hypoxic conditions. Our findings reveal that magnetic associated cell sorting (MACS) does not markedly influence growth characteristics as demonstrated by the generation doubling time. There was, however, an effect regarding an altered adipogenic, osteogenic, and chondrogenic differentiation capacity in the selected cell fraction. In contrast, in the unselected cell population neuronal differentiation is enhanced.
    02/2011; 2011:715341. DOI:10.4061/2011/715341
Show more