Influence of the Tissue Collection Procedure on the Adipogenic Differentiation of Human Stem Cells: Ischemic versus Well-Vascularized Adipose Tissue

Abstract

Clinical and basic science applications using adipose-derived stem cells (ADSCs) are gaining popularity. The current adipose tissue harvesting procedures introduce nonphysiological conditions, which may affect the overall performance of the isolated ADSCs. In this study, we elucidate the differences between ADSCs isolated from adipose tissues harvested within the first 5 min of the initial surgical incision (well-vascularized, nonpremedicated condition) versus those isolated from adipose tissues subjected to medications and deprived of blood supply during elective free flap procedures (ischemic condition). ADSCs isolated from well-vascularized and ischemic tissues positively immunostained for several standard stem cell markers. Interestingly, the percent change in the CD36 expression for ADSCs isolated from ischemic versus well-vascularized tissue was significantly lower in males than females (p < 0.05). Upon differentiation and maturation to adipocytes, spheroids formed using ADSCs isolated from ischemic adipose tissue had lower triglyceride content compared to those formed using ADSCs isolated from the well-vascularized tissue (p < 0.05). These results indicate that ADSCs isolated from ischemic tissue either fail to uptake fatty acids or fail to efficiently convert those fatty acids into triglycerides. Therefore, more robust ADSCs suitable to establish in vitro adipose tissue models can be obtained by harvesting well-vascularized and nonpremedicated adipose tissues.

Keywords: 3D spheroid; CD36; adipose tissue; flow cytometry; triglyceride.

Figure 1

Site of adipose tissue collection during anterolateral thigh (ALT) free flap surgery. Well-vascularized tissue was harvested within the first 5 min of the initial surgical incision while ischemic tissue was harvested from the more distal part of the flaps.

Figure 2

Optical microscopy images of ADSCs isolated from (a) well-vascularized and (b) ischemic adipose tissues. Scale bar = 100 µm.

Figure 3

(a) Representative flow cytometry histograms of ADSCs isolated from well-vascularized and ischemic adipose tissues. (b) Quantitative measurement of the various CD markers. There were no statistically significant differences (p > 0.05) in the CD markers between the ADSCs isolated from the well-vascularized and ischemic tissues.

Figure 4

(a) Quantitative measurement of CD36 marker for ADSCs isolated from well−vascularized and ischemic adipose tissues of male and female patients. (b) The relative change in the CD36 marker for ADSCs isolated from well−vascularized and ischemic adipose tissues of male and female patients. Error bars indicate 95% confidence intervals. * p ≤ 0.05.

Figure 5

Quantitative measurement of various stem CD markers for undifferentiated ADSCs (D0), three-dimensional (3D) spheroid aggregates after 3 days of differentiation (M0) in adipogenic lineage, and ADSCs spheroid differentiated to adipocytes and matured for 10 days (M10). * p ≤ 0.05 compared to D0.

Figure 6

Spheroid formation of ADSCs isolated from the well-vascularized and ischemic tissues atop the ELP-PEI coated surface. (a) Bright field morphology, scale bars = 100 µm. The insets show unilocular fat deposits. (b) Quantitative measurement of the ADSC spheroid sizes (n > 50). Error bars indicate 95% confidence intervals. * p ≤ 0.05 between M0 and M10 timepoints for the same tissue source.

Figure 7

(a) DNA quantification; (b) normalized protein content; (c) normalized triglyceride content of spheroids prepared using the ADSCs isolated from the well-vascularized and ischemic tissues after 3 days of differentiation (Day 0 of maturation, M0) and 10 days of maturation (M10). Error bars represent 95% confidence intervals. * p ≤ 0.05 for M10 versus M0 values of the same group; # p ≤ 0.05 between groups on the same day.