Fas-L promotes the stem cell potency of adipose-derived mesenchymal cells

Abstract

Fas-L is a TNF family member known to trigger cell death. It has recently become evident that Fas-L can transduce also non-apoptotic signals. Mesenchymal stem cells (MSCs) are multipotent cells that are derived from various adult tissues. Although MSCs from different tissues display common properties they also display tissue-specific characteristics. Previous works have demonstrated massive apoptosis following Fas-L treatment of bone marrow-derived MSCs both in vitro and following their administration in vivo. We therefore set to examine Fas-L-induced responses in adipose-derived stem cells (ASCs). Human ASCs were isolated from lipoaspirates and their reactivity to Fas-L treatment was examined. ASCs responded to Fas-L by simultaneous apoptosis and proliferation, which yielded a net doubling of cell quantities and a phenotypic shift, including reduced expression of CD105 and increased expression of CD73, in association with increased bone differentiation potential. Treatment of freshly isolated ASCs led to an increase in large colony forming unit fibroblasts, likely produced by early stem cell progenitor cells. Fas-L-induced apoptosis and proliferation signaling were found to be independent as caspase inhibition attenuated Fas-L-induced apoptosis without impacting proliferation, whereas inhibition of PI3K and MEK, but not of JNK, attenuated Fas-L-dependent proliferation, but not apoptosis. Thus, Fas-L signaling in ASCs leads to their expansion and phenotypic shift toward a more potent stem cell state. We speculate that these reactions ensure the survival of ASC progenitor cells encountering Fas-L-enriched environments during tissue damage and inflammation and may also enhance ASC survival following their administration in vivo.

Fig. 1. Fas-L treatment promotes both apoptosis and proliferation of SVF (P0 ASCs) in culture.

Human SVF cells at P0 were cultured for 14 days, under normal culture conditions or with increasing concentrations of Fas-L. Cell numbers aI and sub-G1 proportions aII and bI and II were calculated by standard cell counts and by propidium iodide (PI) cell cycle flow cytometry analysis, respectively. The experiment was repeated three times using cells from three independent patients. Data are presented as the mean ± standard deviation. bIII Images of cultured P0 ASCs cells with or without 50 ng/ml Fas-L. Floating cells of treated cells (marked by an arrow) were collected and examined using the annexin PI assay. All floating cells were determined to be in different apoptosis stages. c CD95 (Fas) expression on Fas-L treated and untreated cells was examined by Flow cytometry analysis. CD95 demonstrated similar expression levels in Fas-L treated (50 ng/ml) and untreated cells.

Fig. 2. Fas-L treatment leads to an increase in CD105-low and CD73-high subpopulations of cultured SVF cells at passages 0 and 1.

P0 (a) and P1 ASCs (b), cultured under normal culture conditions or with 50 ng/ml Fas-L, were examined for their surface marker expression of CD45, CD31, CD34, CD29, CD105, and CD73 by flow cytometry. The experiment was repeated two times using cells from two independent patients giving the same trend

Fig. 3. Fas-L treatment increases the bone differentiation capacity of cultured SVF cells (P0 ASCs).

Human SVF cells at P0 were cultured for 14 days under normal culture conditions or with 50 ng/ml Fas-L. Treated and untreated cells were than passaged and induced to undergo fat or bone differentiation using designated differentiation media at P1. Differentiation into bone and fat was detected by Alizarin red and Oil red O staining, respectively. Cells differentiated to fat (a) and bone (b) were then photographed and the stain was extracted and quantified. The experiment was repeated three times using cells from three independent patients giving the same trend

Fig. 4. Fas-L treatment of SVF cells increases large CFU-F formation.

Human SVF cells were cultured at low densities, for 21 days, under normal culture conditions or with 50 ng/ml Fas-L. Colonies were then stained by Giemsa and counted. Colonies were photographed (a) and the total number of colonies and of large colonies (>100 cells) was compared between Fas-L-treated and untreated cells b. The experiment was repeated two times using cells from two independent patients giving the same trend

Fig. 5. Fas-L-treated P3 ASCs and Fas-L-treated SVF cells (P0 ASCs) display a similar phenotype.

ASCs (P3) were cultured for 6 days under normal culture conditions or with increasing Fas-L concentrations. Cell numbers (aI) and sub-G1 proportions aII and b were examined by standard cell counts and by propidium iodide (PI) cell cycle flow cytometry analysis, respectively. The experiment was repeated three times using cells from three independent patients. Data are presented as the mean ± standard deviation. P3 ASCs cultured under normal culture conditions or with 50 ng/ml Fas-L, surface marker expression of CD29, CD105, and CD73 was examined by flow cytometry (c)

Fig. 6. Caspase inhibition prevents Fas-L-induced apoptosis but not Fas-L-induced proliferation of ASCs.

ASCs (P2-3) were treated for 4 days with the Fas-L, caspase inhibitor Z-VAD-fmk or both. Sub-G1 proportions a and c and cell numbers (b) were determined by PI cell cycle flow cytometry analysis and standard cell counts, respectively. The experiment was repeated three times using cells from three independent patients. Data are presented as the mean ± standard deviation. d Representative images of ASCs under the different conditions are shown

Fig. 7. Inhibition of PI3K/Akt and MAP kinases ERK-1/2 but not JNK attenuates Fas-L-induced proliferation.

Cultured ASCs (P2-4) were treated with the indicated combination of mega-Fas-L, Z-VAD-fmk, LY294002 (PI3K inhibitor), AZD6244 (ERK-1/2 inhibitor), and SP600125 (JNK inhibitor). Four days later, cells were removed from plates by trypsin and cells were counted using a standard cell counter. Passage 2–4 ASCs were used in all experiments. The experiment was repeated three times using cells from three independent patients. Data are presented as the mean ± standard deviation