Ex Vivo Human Adipose Tissue Derived Mesenchymal Stromal Cells (ASC) Are a Heterogeneous Population That Demonstrate Rapid Culture-Induced Changes

Abstract

Human adipose-derived mesenchymal stromal cells (ASC) are showing clinical promise for the treatment of a range of inflammatory and degenerative conditions. These lipoaspirate-derived cells are part of the abundant and accessible source of heterogeneous stromal vascular fraction (SVF). They are typically isolated and expanded from the SVF via adherent cell culture for at least 2 weeks and as such represent a relatively undefined population of cells. We isolated ex vivo ASC directly from lipoaspirate using a cocktail of antibodies combined with immunomagnetic bead sorting. This method allowed for the rapid enrichment of a defined and untouched ex vivo ASC population (referred to as MACS-derived ASC) that were then compared to culturederived ASC. This comparison found that MACS-derived ASC contain a greater proportion of cells with activity in in vitro differentiation assays. There were also significant differences in the secretion levels of some key paracrine molecules. Moreover, when the MACS-derived ASC were subjected to adherent tissue culture, rapid changes in gene expression were observed. This indicates that culturing cells may alter the clinical utility of these cells. Although MACS-derived ASC are more defined compared to culture-derived ASC, further investigations using a comprehensive multicolor flow cytometry panel revealed that this cell population is more heterogeneous than previously appreciated. Additional studies are therefore required to more precisely delineate phenotypically distinct ASC subsets with the most therapeutic potential. This research highlights the disparity between ex vivo MACS-derived and culture-derived ASC and the need for further characterization.

microRNAs in Ex Vivo Human Adipose Tissue Derived Mesenchymal Stromal Cells (ASC) Undergo Rapid Culture-Induced Changes in Expression, Including miR-378 which Promotes Adipogenesis

“…These ex vivo ASC (herein referred to as ex vivo or MACS-derived ASC, were subjected to a quantitative adipogenic differentiation assay that assesses FABP4 expression by immunohistochemistry. In agreement with our previous report [3], a significantly greater proportion of ex vivo ASC expressed FABP4 in this assay when compared to ex vivo ASC which were cultured for 28 days (day 0 = 57.3%, st. dev. +/− 21.6% versus day 28 = 28.5%, st. dev.…”

“…Our hypothesis was that changes in microRNA expression may underlie the loss of activity that was observed in in vitro differentiation assays that we and others had observed [3,[15][16][17]. The data that are presented here indicate that that is the case, as demonstrated by an over-expression of miR-378 leading to an increase in FABP4 accumulation, which is suggestive of increased differentiation down the adipogenic pathway.…”

“…We previously reported on culture-induced mRNA changes in ex vivo ASC [3]. We were also interested to examine what effect time in culture would have on microRNA expression, as microRNAs are critical regulators of cellular function, including metabolism, homeostasis, proliferation, and cell fate [9].…”

“…We were also interested to examine what effect time in culture would have on microRNA expression, as microRNAs are critical regulators of cellular function, including metabolism, homeostasis, proliferation, and cell fate [9]. To this end, we used immunomagnetic bead sorting to isolate ex vivo, untouched ASC from three donors while using a cocktail of FITC-labelled antibodies (anti-CD31, CD45, CD146, and CD235a) designed to remove all non-ASC from the SVF, as described previously [3]. Flow cytometry analyses indicated that the post sort purity of the enriched fraction was > 97% for CD34+, CD73+, and CD90+ (data not shown).…”

“…We previously reported a method that allowed for the rapid enrichment of a defined and untouched ex vivo ASC population that were then compared to culture-derived ASC [3]. This comparison found that ex vivo ASC demonstrate rapid changes in mRNA expression when exposed to adherent-tissue culture that are likely to effect the function and potency of these cells.…”