CD34+CD146+ adipose-derived stromal cells enhance engraftment of transplanted fat

Abstract

Fat grafting is a surgical technique able to reconstruct and regenerate soft tissue. The adipose-derived stromal cells (ASCs) within the stromal vascular fraction are believed to drive these beneficial effects. ASCs are increasingly recognized to be a heterogeneous group, comprised of multiple stem and progenitor subpopulations with distinct functions. We hypothesized the existence of an ASC subpopulation with enhanced angiogenic potential. Human ASCs that were CD34+CD146+, CD34+CD146−, or CD34+ unfractionated (UF) were isolated by flow cytometry for comparison of expression of proangiogenic factors and endothelial tube-forming potential. Next, lipoaspirate was enriched with either CD34+CD146+, CD34+CD146−, CD34+ UF ASCs, or was not enriched, and grafted beneath the scalp skin of immunodeficient CD-1 Nude mice (10 000 cells/200 μL/graft). Fat retention was monitored radiographically more than 8 weeks and fat grafts were harvested for histological assessment of quality and vascularization. The CD34+CD146+ subpopulation comprised 30% of ASCs, and exhibited increased expression of vascular endothelial growth factor and angiopoietin-1 compared to CD34+CD146− and CD34+ UF ASCs, and increased expression of fibroblast growth factor-2 compared to CD34+CD146− ASCs. The CD34+CD146+ subpopulation exhibited enhanced induction of tube-formation compared to CD34+CD146− ASCs. Upon transplantation, fat enriched CD34 +CD146+ ASCs underwent less resorption and had improved histologic quality and vascularization. We have identified a subpopulation of CD34+ ASCs with enhanced angiogenic effects in vitro and in vivo, likely mediated by increased expression of potent proangiogenic factors. These findings suggest that enriching lipoaspirate with CD34+CD146+ ASCs may enhance fat graft vascularization and retention in the clinical setting.

Water jet-assisted lipoaspiration and Sepax cell separation system for the isolation of adipose stem cells with high adipogenic potential

“…Another study also reported a tendency for stronger adipogenic potential of cells isolated with water jet-assisted liposuction versus cells obtained by suction-assisted liposuction [12] . We have also described that CD146 + ASCs have enhanced angiogenic potential [10] , which was confirmed by another recent study that showed the benefits of supplementing fat grafts with CD146-selected cells [16] . We found that there was a tendency for higher in vitro angiogenesis evoked by water jet-isolated cells and higher secretion of VEGF-A.…”

A comparative analysis of deferoxamine treatment modalities for dermal radiation‐induced fibrosis

“…Incubation was performed with anti‐CD31 primary antibody (1:100, ab28364; Abcam) followed by an Alexa Fluor 647‐conjugated donkey anti‐rabbit IgG secondary antibody (1:500, ab150075; Abcam). Red pixel area was obtained from 20× magnification images ( n = 15 per condition) via ImageJ (NIH) analysis that recognized red hues, binarized the images and counted selected pixels 31 …”

Angiogenic CD34+CD146+ adipose‐derived stromal cells augment recovery of soft tissue after radiotherapy

“…Adipose‐derived stromal cells were sorted out of the resuspended pellet from liposuction in FACS buffer and stained with antibodies for human reactivity: CD45‐Pacific blue (BioLegend, Cat#304022) to indicate hematopoietic cells; CD235a (Glycophorin A)‐eFluor 450 (BioLegend, Cat#349108) to indicate erythrocytes, and erythroid progenitors; CD31(PECAM‐1 or platelet endothelial cell adhesion molecule)‐eFluor 450 (BioLegend, Cat#303114) to indicate endothelial cells; CD34‐AlexaFluor‐488 (BioLegend, Cat#343517); and CD146‐PE/Cy7 (BioLegend, Cat#361007). To isolate the ASC subpopulations of interest, flow cytometry (FACS Aria II, BD Biosciences) was performed to create a Lineage‐negative (CD45‐CD235a‐CD31) and a positive gating strategy: CD34+CD146+, CD34+CD146−, and unfractionated (UF) CD34+ ASCs (Figure 2) (Borrelli, Patel, Blackshear, et al., 2020). Purity was tested prior ASC subpopulation sorting via analysis of a 2000 fresh cell sort that resulted in >99% purity for the three subpopulations of interest.…”

“…Recently, we described a stromal cell subpopulation within SVF cells positive for CD34 and CD146 with increased expression of vascular endothelial growth factor (VEGF) and angiopoietin (ANGPT)‐1 (Borrelli, Patel, Blackshear, et al., 2020). These cells promoted vessel formation within human fat grafts placed into mice and ultimately increased graft retention (Borrelli, Patel, Blackshear, et al., 2020). However, this study only observed CD146+ ASCs within fat grafts in nonirradiated mice.…”