Prospective purification of perivascular presumptive mesenchymal stem cells from human adipose tissue: process optimization and cell population metrics across a large cohort of diverse demographics.

C C West,K Sukhija,K Ting,S E Lobo,C Soo,D C Hay,M Corselli,K L March,A W James,C Black,I R Murray,W R Hardy,V Lagishetty,P Liang,B Péault,S Pang

doi:10.1186/s13287-016-0302-7

Abstract

BackgroundAdipose tissue is an attractive source of mesenchymal stem cells (MSC) as it is largely dispensable and readily accessible through minimally invasive procedures such as liposuction. Until recently MSC could only be isolated in a process involving ex-vivo culture and their in-vivo identity, location and frequency remained elusive. We have documented that pericytes (CD45-, CD146+, and CD34-) and adventitial cells (CD45-, CD146-, CD34+) (collectively termed perivascular stem cells or PSC) represent native ancestors of the MSC, and can be prospectively purified using fluorescence activated cell sorting (FACS). In this study we describe an optimized protocol that aims to deliver pure, viable and consistent yields of PSC from adipose tissue. We analysed the frequency of PSC within adipose tissue, and the effect of patient and procedure based variables on this yield.MethodsWithin this twin centre study we analysed the adipose tissue of n = 131 donors using flow cytometry to determine the frequency of PSC and correlate this with demographic and processing data such as age, sex, BMI and cold storage time of the tissue.ResultsThe mean number of stromal vascular fraction (SVF) cells from 100 ml of lipoaspirate was 34.4 million. Within the SVF, mean cell viability was 83 %, with 31.6 % of cells being haematopoietic (CD45+). Adventitial cells and pericytes represented 33.0 % and 8 % of SVF cells respectively. Therefore, a 200 ml lipoaspirate would theoretically yield 23.2 million viable prospectively purified PSC - sufficient for many reconstructive and regenerative applications. Minimal changes were observed in respect to age, sex and BMI suggesting universal potential application.ConclusionsAdipose tissue contains two anatomically and phenotypically discreet populations of MSC precursors – adventitial cells and pericytes – together referred to as perivascular stem cells (PSC). More than 9 million PSC per 100 ml of lipoaspirate can be rapidly purified to homogeneity using flow cytometry in clinically relevant numbers potentially circumventing the need for purification and expansion by culture prior to clinical use. The number and viability of PSC are minimally affected by patient age, sex, BMI or the storage time of the tissue, but the quality and consistency of yield can be significantly influenced by procedure based variables.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0302-7) contains supplementary material, which is available to authorized users.

Highlights

Adipose tissue is an attractive source of mesenchymal stem cells (MSC) as it is largely dispensable and readily accessible through minimally invasive procedures such as liposuction
More than 9 million perivascular stem cells (PSC) per 100 ml of lipoaspirate can be rapidly purified to homogeneity using flow cytometry in clinically relevant numbers potentially circumventing the need for purification and expansion by culture prior to clinical use
The number and viability of PSC are minimally affected by patient age, sex, Body mass index (BMI) or the storage time of the tissue, but the quality and consistency of yield can be significantly influenced by procedure based variables

Summary

Introduction

Adipose tissue is an attractive source of mesenchymal stem cells (MSC) as it is largely dispensable and readily accessible through minimally invasive procedures such as liposuction. In a series of pioneering experiments in the 1960s, Alexander Friedenstein et al [1] identified a population of cells from rodent bone marrow that adhered to culture vessels, formed colonies, could differentiate into osteoblasts in culture, and generated bone when implanted ectopically in vivo [2, 3] Friedenstein termed these cells colony forming unit fibroblast (CFU-F), until Arnold Caplan [4], in the early 1990s, coined the term mesenchymal stem cells (MSC). MSC have been enlisted from bone marrow and multiple other tissues, according to their ability to adhere and grow on plastic [6] This suggested that these cells possess a common identity and a widespread anatomical distribution but provided little insight into location, cellular phenotype, frequency, and specific properties of these cells. Some of these questions were answered when Crisan et al [7] demonstrated that microvascular pericytes in multiple human fetal and adult tissues express MSC markers, and that when purified to homogeneity by fluorescence-activated cell sorting (FACS) and cultured they are identical to conventional MSCs in terms of morphology, phenotype, and function

Methods

Results

Discussion

Conclusion