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

Abstract

Background: 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. 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.

Methods: Within 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.

Results: The 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.

Conclusions: Adipose 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.

Keywords: Adipose tissue; Adipose-derived stem cell; Cell sorting; Flow cytometry; Mesenchymal stem cells; Pericyte; Tunica adventitia.

Fig. 1

Gating strategies for the isolation of PSC from the SVF. Gate 1 = cells, Gate 2 = single cells, Gate 3 = live cells, Gate 4 = CD45⁻ (nonhaematopoietic cells), Gate 5 = CD31^– (nonendothelial cells), Gate ADV = adventitial cells (CD31⁻, CD45⁻, CD34⁺, CD146⁻), and Gate PERI = pericytes (CD31⁻, CD45⁻, CD34⁺, CD146⁻). DAPI 4',6-diamidino-2-phenylindole, FSC forward scatter, SSC side scatter

Fig. 2

a, b. Linear fit of SVF yield and PSC yield (× 10⁶) per 100 ml of lipoaspirate with respect to donor age (n = 124, R = 0.07 and 0.09 respectively). c, d. One-way analysis of variance of SVF and PSC yields (× 10⁶) vs donor gender (n = 131; male = 19, female = 112): green diamonds reflect the mean yield (center line) and 95 % confidence interval (vertical span) for each gender, and the grand mean in gray. Linear fit of SVF yield e and proportion of PSCs comprising the SVF f with respect to donor BMI (n = 97, R = 0.05 and 0.01 respectively). BMI body mass index, F female, lipo lipoaspirate, M male, PSC perivascular stem cell, SVF stromal vascular fraction (Color figure online)

Fig. 3

One-way analysis of variance analysis of the number and proportion of cells recovered with respect to storage time at 4 °C. a PSC yield (× 10⁶) per 100 ml of lipoaspirate showing a gradual increase with longer storage times; however, this is a relative effect due to the deceasing number of CD45⁺ hematopoietic cells seen in b. Green diamonds indicate the mean and 95 % confidence interval for each storage time interval, while the gray line represents the grand or overall mean. Hrs hours, PSC perivascular stem cell, SVF stromal vascular fraction (Color figure online)

Fig. 4

Statistical control chart demonstrating that optimization improved the reproducibility of PSC isolation and confirming that both the UCLA and UK isolation processes are under statistical control. A Levey-Jennings chart depicting individual data points for PSC yield (×10⁶) obtained from 100 ml of lipoaspirate using the UCLA isolation process, before and after optimization, as compared with the UK process (n = 131). The central green line represents the general mean and is delimited by upper and lower control limits (red lines) based upon a 3σ interval. PSC perivascular stem cell, SD standard deviation, SVF stromal vascular fraction, UCLA University of California at Los Angeles (Color figure online)

Fig. 5

Improvements in PSC purity. a Optimization of the PSC isolation process has led to increased purity of cells, as indicated by enrichment for the adventitial cell antigen CD34 (left), and the dramatic reduction in the endothelial cell antigen CD31 (middle) and haematopoietic marker CD45 (right) by real-time quantitative PCR (n = 7; pre optimization = 4, post optimization = 3). b (Left) FSC vs SSC demonstrating the population of lymphocytes (arrow). (Centre) Confirmation of lymphocytes by demonstration of CD45⁺ phenotype of the subpopulation. (Right) Selection of the CD45^– depleted cellular fraction for subsequent analysis. FSC Forward scatter, PSC perivascular stem cell, SSC side scatter