Deciphering influence of donor age on adipose-derived stem cells: in vitro paracrine function and angiogenic potential

Abstract

Background: As fat grafting is commonly used as a filler, Adipose-derived stem/stromal cells (ASC) have been reported to be key player in retention rate. Paracrine and differentiation potential of those cells confer them strong pro-angiogenic capacities. However, a full characterization of the influence of aging on ASC has not been reported yet. Here we've investigated the effect of age on paracrine function, stemness and angiogenic potential of ASC.

Methods: ASC were extracted from young and old adult donors. We assessed stromal vascular fraction cell populations repartition, ASC stemness potential, capability to differentiate into mesenchymal lineages as well as their secretome. Angiogenic potential was assessed using a sprouting assay, an indirect co-culture of ASC and dermal microvascular endothelial cells (EC). Total vascular sprout length was measured, and co-culture soluble factors were quantified. Pro-angiogenic factors alone or in combination as well as ASC-conditioned medium (CM) were added to EC to assess sprouting induction.

Results: Decrease of endothelial cells yield and percentage is observed in cells extracted from adipose tissue of older patients, whereas ASC percentage increased with age. Clonogenic potential of ASC is stable with age. ASC can differentiate into adipocytes, chondrocytes and osteoblasts, and aging does not alter this potential. Among the 25 analytes quantified, high levels of pro-angiogenic factors were found, but none is significantly modulated with age. ASC induce a significantly longer vascular sprouts compared to fibroblasts, and no difference was found between young and old ASC donors on that parameter. Higher concentrations of FGF-2, G-CSF, HGF and IL-8, and lower concentrations of VEGF-C were quantified in EC/ASC co-cultures compared to EC/fibroblasts co-cultures. EC/ASC from young donors secrete higher levels of VEGF-A compared to old ones. Neither soluble factor nor CM without cells are able to induce organized sprouts, highlighting the requirement of cell communication for sprouting. CM produced by ASC supporting development of long vascular sprouts promote sprouting in co-cultures that establish shorter sprouts.

Conclusion: Our results show cells from young and old donors exhibit no difference in all assessed parameters, suggesting all patients could be included in clinical applications. We emphasized the leading role of ASC in angiogenesis, without impairment with age, where secretome is a key but not sufficient actor.

Keywords: Adipose-derived stem/stromal cells; Aging; Angiogenesis; Fat graft; Paracrine.

Fig. 1

Stromal vascular fraction was analyzed with flow cytometry and then ASC were cultured in order to assess their clonogenic, chondrogenic, osteogenic, adipogenic and pro-angiogenic potentials as well as their secretome. (Created with BioRender)

Fig. 2

Colony forming efficiency of ASC from young and old donors. (A) photography of CFE from young and old donors, 2 pictures of the same well are presented for each donor. Original magnification 10x. (B) quantification of colonies formed by ASC after 15 days of culture. n = 6 donors were used in each age category. No significant difference was found between CFE of ASC from young and old donors. Unpaired Student t-test was used to test the significance.

Fig. 3

ASC differentiation into lineages. (A) Microscopic images of ASC at P4 in proliferation medium (control) or differentiation medium (differentiated) for 3 lineages. Adipocytes were stained with Bodipy probe and Hoechst to visualize neutral lipid droplets and nucleus, chondrocytes were stained with Alcian blue to visualize proteoglycans and osteoblasts were stained with either BCIP/NBT or Alizarin Red S to visualize alkaline phosphatase (early differentiation) or mineralization (late differentiation) respectively (original magnification 10x). Bodipy (B) or ARS (C) quantification of control or differentiated ASC according to age category. n = 6 donors were used in each age category. Paired Wilcoxon test was used to test the significance. * P value < 0,05.

Fig. 4

Secretome of ASC according to age category. n = 6 donors were used in each age category. Unpaired Wilcoxon test was used to test the significance. * P value < 0,05.

Fig. 5

(A) Diversity of sprouting of endothelial cells from dextran beads stained with phalloidin and Hoechst to visualize actin and nucleus, respectively (original magnification 10x, bar, 200 μm). (B) Total sprouts length with co-culture of EC with ASC (EC/ASC; dark grey) and fibroblasts (EC/Fb; light grey) as stimulating cells. (B) Total sprouts length with co-culture of EC with either ASC (EC/ASC; dark grey) and fibroblasts (EC/Fb; light grey) as stimulating cells. (C) Total sprouts length according to co-culture of EC with ASC from young (EC/ASC y; blue) or old (EC/ASC o; red) donors as stimulating cells. n = 5 donors for ASC young, n = 6 donors for ASC old, n = 1 donor for fibroblasts, experiments were conducted in 4 replicates. Unpaired Wilcoxon test was used to test the significance. * P value < 0.05.

Fig. 6

(A) Secretome of EC without stimulating cells (EC; clear box), or EC co-culture with either ASC (EC/ASC; dark grey) (independently of donor age) or fibroblasts (EC/Fb; light grey). (B) Secretome of EC co-culture with ASC from young (EC/ASC y; blue) or old (EC/ASC o; red) donor. n = 5 donors for ASC young, n = 6 donors for ASC old, n = 1 donor for fibroblasts, experiments were conducted in 4 replicates. ANOVA test was used to test the significance in (A). Unpaired Wilcoxon test was used to test the significance in (B). * P value < 0,05.

Fig. 7

EC required cells and specific dosage of soluble factors to form organized. (A) Confocal images of sprouting beads according to conditions. (B) Total sprout length quantification (normalized cells alone) supplemented with ASC-CM. ASC donor is specified in the graph. Experiments were conducted in duplicate. ANOVA test was used to test the significance. * P value < 0,05.