Phenotypic Analysis of Stromal Vascular Fraction after Mechanical Shear Reveals Stress-Induced Progenitor Populations

Abstract

Background: Optimization of fat grafting continues to gain increasing attention in the field of regenerative medicine. "Nanofat grafting" implements mechanical emulsification and injection of standard lipoaspirate for the correction of superficial rhytides and skin discoloration; however, little is known about the cellular constituents of the graft. Based on recent evidence that various stressors can induce progenitor activity, the authors hypothesized that the shear forces used in common fat grafting techniques may impact their regenerative capacities.

Methods: Lipoaspirates were obtained from 10 patients undergoing elective procedures. Half of each sample was subjected to nanofat processing; the other half was left unchallenged. The viscosity of each sample was measured for computational analysis. The stromal vascular fraction of each sample was isolated, quantified, and analyzed by means of flow cytometry with two multicolor fluorescence antibody panels.

Results: Standard lipoaspirate is ideally suited for mechanical stress induction. The mechanical emulsification involved in nanofat processing did not affect cell number; however, viability was greatly reduced when compared with the stromal vascular fraction of standard lipoaspirate. Interestingly, nanofat processing resulted in stress-induced stromal vascular fraction with a higher proportion of endothelial progenitor cells, mesenchymal stem cells, and multilineage differentiating stress-enduring cells. Single-parameter analysis also revealed significant increases in CD34, CD13, CD73, and CD146 of the stress-induced stromal vascular fraction, markers associated with mesenchymal stem cell activity.

Conclusions: Mechanical processing used in techniques such as nanofat grafting induces the up-regulation of progenitor phenotypes consistent with multipotency and pluripotency. These data provide a first step in characterizing the potential regenerative benefits realized through stress induction in fat grafting.

Clincal question/level of evidence: Therapeutic, V.

Fig. 1

Relationship of kinematic viscosity (bars) and shear force (line) anticipated during the emulsification process of standard lipoaspirate into nanofat. As viscosity decreases, so does the ability to generate shear stress.

Fig. 2

Gating strategy and viability analysis. (Left) A wide gating strategy was employed to capture data on all cells of the stromal vascular fraction by using FSC (forward scatter, representing cell size) and SSC (side scatter representing granularity). (Right) Absolute cell count (propidium iodide labeling) revealed significant decrease in viable cells recovered from lipoaspirate processed with mechanical shear (p = 0.004).

Fig. 3

Flow cytometric comparison of cell phenotypes and summarized data. Histograms representing phenotypic analysis of stromal vascular fraction (SVF) from standard lipoaspirate (Left column) and stress-induced SVF (right column). (Top graph) CD34, a universal stem cell marker, found to be significantly greater in siSVF compared to control (p = 0.001). (Middle graph) Mesenchymal stem cell markers CD13 (p = 0.003), CD73 (p = 0.003) and CD146 (p = 0.03) all found to be greater in siSVF compared to control. (Bottom graph) CD45 (p = 0.004) and CD31 (p = 0.018) were also found to be significantly upregulated in siSVF compared to control.

Fig. 4

Flow cytometric comparison of mesenchymal stem cells (CD45⁻/CD34⁺) and summarized data. (left) Dot plot histogram representing significant upregulation of MSC population in stress-induced SVF (siSVF) when compared to control (red arrow). (Right) Summarized data representing almost 3-fold increase in MSC population (p = 0.006)

Fig. 5

Flow cytometric comparison of stromal vascular fraction subpopulations in standard lipoaspirate: endothelial progenitor cells (EPCs, p = 0.025), pre-adipocytes (p = 0.205), transitional cells (p = 0.237), adipose-derived stem cells (ADSCs, p = 0.024), and pericytes (p = 0.230).

Fig. 6

Flow cytometric comparison of Muse cells (CD13⁺/SSEA-3⁺) and summarized data. (left) Dot plot histogram representing upregulation of Muse population (red box). (Right) Summarized data representing ~3-fold increase in Muse population (p = 0.046).