Awakened by Cellular Stress：Isolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue（3）
Muse-ATs Isolated from Lipoaspirated Human Adipose Tissue
Adipose tissue is composed of adipocytes (mature cells) and the stromal vascular fraction (SVF) containing a heterogeneous population of cells, including adipose tissue macrophages (ATMs), adipose stem cells (ASCs), mesenchymal stem cells, and fibroblasts.
We explored the possibility of both activating and isolating Muse-AT cells from their quiescent state by exposing them to cellular stress (Fig. 1A). Lipoaspirated material was first incubated in collagenase for 30 min at 37°C to release adipocytes (floating cells) and different cellular components present in the SVF as previously described. This material was then subjected to severe cellular stress, including long incubation with collagenase, low temperatures, low serum and hypoxia, to kill fragile adipose cells and release Muse-AT cells. Optimal conditions for the release of Muse-AT cells were determined to be 16 hours incubation with collagenase in DMEM medium without FCS at 4°C under very low O2, which subsequently gave way to a homogenous population of Muse-AT cells. Approximately 90% of isolated cells were both SSEA3 and CD105 positive, as determined by flow cytometry (Fig. 1B). This high purity is presumably due to the severity of the cellular stress conditions, responsible for the depletion of other cell types. As all other components of the adipose tissue lipoaspirate failed to survive, a population of highly purified Muse-AT cells was obtained, and therefore further purification processes were not necessary. Muse-AT cells were plated in both adherent and non-adherent cell culture dishes. We observed that Muse-AT cells can grow either in suspension or in adherence culture to form the characteristic cell clusters observed in ES cell-derived embryoid body, as described in bone marrow and dermal fibroblast-derived Muse cells in previous reports (Fig. 1C, D). Under both conditions, individual Muse-AT cells reached a diameter of around 10µm and cell clusters reached a diameter of up to 50µm by day 3 (Fig. 1C–D), which has been previously demonstrated to mark the limit of their proliferative capacity.
Figure 1. Isolation and morphologic characterization of Muse-ATs.
(A) Schematic of Muse-AT isolation and activation from their quiescent state by exposure to cellular stress. Muse-AT cells were obtained after 16 hours, with incubation with collagenase in DMEM medium without FCS at 4°C under very low O2 (See Methods). (B) FACS analysis demonstrates that 90% of isolated cells are both SSEA3 and CD105 positive. (C) Muse-AT cells can grow in suspension, forming spheres or cell clusters as well as individual cells (see red arrows) or (D) Muse-AT cells can adhere to the dish and form cell aggregates. Under both conditions, individual Muse-AT cells reached a diameter of approximately 10µm and cell clusters reached a diameter of up to 50µm, correlating to stem cell proliferative size capacity.
Muse-ATs Spontaneously Express Pluripotent Stem Cell Markers
Upon transfer and adherence to chamber slides for immunofluorescent staining, both the Muse-AT cell clusters and individual Muse-AT cells strongly expressed all of the characteristic pluripotent stem cell markers that were examined. These included SSEA3, a cell-surface glycosphingolipid frequently used to detect human ES cells and to purify Muse cells from bone marrow and dermis; Oct3/4 a protein involved in the self-renewal of human ES cells; Nanog, a transcription factor involved in the self-renewal of human ES cells; Sox2, a transcription factor that controls genes involved in embryonic development; and TRA-1-60, which reacts with the antigen TRA-1-60 on the surface of embryonic germ cells and ES cells (Fig. 2). Comparatively, ASCs derived from the same lipoaspirated tissue were either negative or weakly positive for these pluripotent stem cell markers (Fig. 2).
Figure 2. Muse-ATs express pluripotent stem cell markers.
Immunofluorescence microscopy demonstrates that Muse-AT aggregates, along with individual Muse-AT cells, express characteristic pluripotent stem cell markers, including SSEA3, Oct3/4, Nanog, Sox2, and TRA1-60. Comparatively, ASCs (right panel) derived from the same lipoaspirate under standard conditions (see above, were negative for these pluripotent stem cell markers. Nuclei were stained with DAPI (blue). Original magnification, 600 X.