Impaired expansion and multipotentiality of adult stromal cells in a rat chronic alcohol abuse model

Abstract

It is well established that bone maintenance and healing is compromised in alcoholics. Adult bone marrow-derived stromal cells (BMSCs) and adipose tissue-derived stromal cells (ASCs) likely contribute to bone homeostasis and formation. Direct and indirect alcohol exposure inhibits osteoprogenitor cell function through a variety of proposed mechanisms. The goal of this study was to characterize the effects of chronic alcohol ingestion on the native number and in vitro growth characteristics and multipotentiality of adult BMSCs and ASCs in a rat model. Adult male Sprague-Dawley rats received a liquid diet containing 36% ethanol or an isocaloric substitution of dextramaltose (control). After 4, 8, or 12 weeks of the diet, ASCs were harvested from epididymal adipose tissue and BMSCs from femoral and tibial bone marrow. Cell doublings (CDs) per day and doubling times (DTs) were determined for primary cells (P0) and cell passages 1 through 6 (P1-P6). Fibroblastic (CFU-F), adipogenic (CFU-Ad), and osteogenic (CFU-Ob) colony-forming unit (CFU) frequencies were assessed for P0, P3, and P6. The CDs and DTs were lower and higher, respectively, for ASCs and BMSCs harvested from ethanol versus control rats at all time points. The CFU-F, CFU-Ad, and CFU-Ob were significantly higher in ASCs harvested from control versus ethanol rats for P0, P3, and P6 at all times. Both CFU-Ad and CFU-Ob were significantly higher in P0 BMSCs harvested from control versus ethanol rats after 12 weeks of the diet. The CFU-Ob for P3 BMSCs from control rats was significantly higher than those from ethanol rats after 8 and 12 weeks on the diet. All three CFU frequencies in ASCs from ethanol rats tended to decrease with increasing diet duration. The ASC cell and colony morphology was different between control and ethanol cohorts in culture. These results emphasize the significant detrimental effects of chronic alcohol ingestion on the in vitro expansion and multipotentiality of adult mesenchymal stromal cells (MSCs). Maintenance of the effects through multiple cell passages in vitro suggests cells may be permanently compromised.

Figure 1

Mean ± SEM rat weight (A) and epididymal adipose weight (B) after 4, 8, or 12 weeks of a 37% EtOH or isocaloric liquid diet. * = Significant difference between diet cohorts within time points.

Figure 2

Number of nucleated cells (mean ± SEM) harvested from epididymal adipose (A) or combined femoral and tibial bone marrow (B) after 4, 8, or 12 weeks of a 37% EtOH or isocaloric liquid diet. * = Significant difference between diet cohorts within time points.

Figure 3

Mean ± doubling time and cell doublings/day for ASCs (A, C) and BMSCs (B, D) after 4, 8, or 12 weeks of a 37% EtOH or isocaloric liquid diet. * = Significant difference between diet cohorts within time points.

Figure 4

Photomicrographs depicting different morphology between P0 ASCs harvested from control (A) versus EtOH (B) rats. The P0 adipose isolates from control rats at each time point were heterogenous, displaying both an elongated, spindle like morphology characteristic of a fibroblast phenotype and a flattened, polygonal shape with short or absent processes. The P0 adipose isolate from EtOH rats had less heterogenous P0 populations, and the majority of cells had a flattened polygonal shape.

Figure 5

Photomicrographs of ASCs harvested from control (left) and EtOH rats (right) following culture in stromal (A,B), adipogenic (C,D), or osteogenic (E,F) media. Colonies from EtOH ASCs were smaller and less organized than those from control ASCs. Staining toluidine blue (A,B), Oil Red O (C,D), Alizarin Red (E,F).