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 culture-derived 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.
Highlights
Human adipose tissue derived stromal cells (ASC) are currently being tested as cell-based therapies against a wide range of diseases and conditions in numerous clinical trials (Bateman et al, 2018) including wound healing, (Bertozzi et al, 2017) cardiovascular disease (Ma et al, 2017) and cartilage regeneration (Pak et al, 2018)
Our previous analysis of the stromal vascular fraction (SVF) from human adipose tissue using multicolor flow cytometry gave us a good understanding of the various cell types present in this tissue (Feisst et al, 2014). Based on this earlier work we used a flow cytometry panel to demonstrate that the ASC population, positive for CD90, CD73, and CD34, could be enriched by excluding all populations positive for CD45, CD235a, FIGURE 1 | Continued
We sought to use a cocktail of FITC-labeled antibodies (CD31, CD45, CD146, and CD235a) and anti-FITC labeled magnetic MACSTM beads to enable enrichment of an “untouched” ASC cell population from the SVF using immunomagnetic column-based sorting
Summary
Human adipose tissue derived stromal cells (ASC) are currently being tested as cell-based therapies against a wide range of diseases and conditions in numerous clinical trials (Bateman et al, 2018) including wound healing, (Bertozzi et al, 2017) cardiovascular disease (Ma et al, 2017) and cartilage regeneration (Pak et al, 2018). The cellular diversity of this population, referred to hereafter as adipose tissue derived mesenchymal stromal cells (ASC), and their true therapeutic potential remains unclear. They are defined in part as being able to differentiate into fat, bone, and cartilage lineages in vitro (Bourin et al, 2013). They are reported to function as bioreactors producing molecules that promote healing and inhibit over activity of the immune system (Ma et al, 2014). Their exact therapeutic mode of action in vivo is unclear (Robey, 2017) increasing evidence points to mesenchymal cells exerting a paracrine effect (Zwolanek et al, 2017; Caplan, 2019) rather than cell replacement
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