Abstract
Human adipose tissue is a rich source of adipose-derived stem cells (ASCs) and vascular endothelial progenitor cells (EPCs). However, no standardized method has been established for the isolation and purification of adipose-derived EPCs (AEPCs). The aim of this study was to establish a method for the isolation and purification of AEPCs. The stromal vascular fraction (SVF) was extracted from human lipoaspirates, and the CD45−CD31+ fraction of the SVF was collected by magnetic-activated cell sorting (MACS). The CD45−CD31+ fraction was cultured for 4.5 days, followed by a second MACS separation to collect the CD31+ fraction. Purified AEPCs were expanded without being overwhelmed by proliferating ASCs, indicating that a high level (> 95%) of AEPC purification is a key factor for their successful isolation and expansion. AEPCs exhibited typical endothelial markers, including CD31, von Willebrand factor, and the isolectin-B4 binding capacity. AEPCs formed colonies, comparable to cultured human umbilical vein endothelial cells (HUVECs). Both AEPCs and HUVECs formed capillary-like networks in the tube formation assay, with no significant difference in network lengths. We are the first to establish a purification and expansion method to isolate these cells. Because adipose tissue is a clinically accessible and abundant tissue, AEPCs may have potential advantages as a therapeutic tool for regenerative medicine.
Highlights
Human adipose tissue is a rich source of adipose-derived stem cells (ASCs) and vascular endothelial progenitor cells (EPCs)
The stromal vascular fraction (SVF) could be segregated into four primary cell populations, adipose-derived EPCs (AEPCs) (CD45−CD34+CD31+), ASCs (CD45−CD34+CD31−), hematopoietic cells (CD45+), and other cell types (CD45−CD34−; Fig. 1B)
Fresh AEPC population was further analyzed for other surface markers and characterized as CD45−CD31+CD34+CD105+CD146+CD157±CD200±
Summary
Human adipose tissue is a rich source of adipose-derived stem cells (ASCs) and vascular endothelial progenitor cells (EPCs). The aim of this study was to establish a method for the isolation and purification of AEPCs. The stromal vascular fraction (SVF) was extracted from human lipoaspirates, and the CD45−CD31+ fraction of the SVF was collected by magnetic-activated cell sorting (MACS). Abbreviations EPCs Endothelial progenitor cells ECs Endothelial cells IRB Institutional review board SVF Stromal vascular fraction AEPCs Adipose-resident microvascular endothelial progenitor cells DNase[1] Deoxyribonuclease 1 Pol[188] Poloxamer 188 HBSS Hanks’ balanced salt solution MACS Magnetic-activated cell sorting FBS Fetal bovine serum FVD780 Fixable viability dye eFluor 780 HUVECs Human umbilical vein endothelial cells FACS Fluorescence-activated cell sorting BSA Bovine serum albumin EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid DPBS Dulbecco’s phosphate-buffered saline RT Room temperature vWF Von Willebrand factor DAPI 4′,6-Diamidino-2-phenylindole PFA Paraformaldehyde in phosphate buffer solution.
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