title: 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.
authors.affiliation: British Heart Foundation Centre for Vascular Regeneration & Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.|Department of Plastic and Reconstructive Surgery, St Johns Hospital, Howden Road West, Livingston, UK.
authors.forename: C C
authors.initials: CC
authors.lastname: West
authors.affiliation: Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, USA.
authors.forename: W R
authors.initials: WR
authors.lastname: Hardy
authors.affiliation: British Heart Foundation Centre for Vascular Regeneration & Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: I R
authors.initials: IR
authors.lastname: Murray
authors.affiliation: Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, USA.
authors.forename: A W
authors.initials: AW
authors.lastname: James
authors.affiliation: Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, USA.|BD Biosciences, San Diego, CA, USA.
authors.forename: M
authors.initials: M
authors.lastname: Corselli
authors.affiliation: Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, USA.
authors.forename: S
authors.initials: S
authors.lastname: Pang
authors.affiliation: Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, USA.|Bone and Joint Research Group, Institute of Developmental Sciences, University of Southampton, Southampton, UK.
authors.forename: C
authors.initials: C
authors.lastname: Black
authors.affiliation: Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, USA.|Department of Surgery, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
authors.forename: S E
authors.initials: SE
authors.lastname: Lobo
authors.affiliation: Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, USA.|Department of Emergency Medicine, Kaweah Delta Health Care District, Visalia, CA, USA.
authors.forename: K
authors.initials: K
authors.lastname: Sukhija
authors.affiliation: Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, USA.|Department of Urology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA.
authors.forename: P
authors.initials: P
authors.lastname: Liang
authors.affiliation: Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, USA.|Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA.
authors.forename: V
authors.initials: V
authors.lastname: Lagishetty
authors.affiliation: British Heart Foundation Centre for Vascular Regeneration & Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: D C
authors.initials: DC
authors.lastname: Hay
authors.affiliation: Indiana Center for Vascular Biology and Medicine, Krannert Institute of Cardiology, and Vascular and Cardiac Center for Adult Stem Cell Research, Indiana University, Bloomington, IN, USA.
authors.forename: K L
authors.initials: KL
authors.lastname: March
authors.affiliation: Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, USA.|Division of Growth and Development and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, CA, 90095, USA.
authors.forename: K
authors.initials: K
authors.lastname: Ting
authors.affiliation: Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, USA.|Division of Plastic and Reconstructive Surgery, Department of Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, 90095, USA.|Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, 90095, USA.
authors.forename: C
authors.initials: C
authors.lastname: Soo
authors.affiliation: British Heart Foundation Centre for Vascular Regeneration & Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK. bpeault@mednet.ucla.edu.|Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, CA, USA. bpeault@mednet.ucla.edu.
authors.forename: B
authors.initials: B
authors.lastname: Péault
mesh_terms: D000328:Adult; D015703:Antigens, CD / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D003710:Demography; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D008875:Middle Aged; D020286:Pericytes / Q000378:metabolism; D011446:Prospective Studies; D050151:Subcutaneous Fat / Q000166:cytology; D014021:Tissue Preservation; D055815:Young Adult
chemical_list: D015703:Antigens, CD
keywords: Adipose tissue; Adipose-derived stem cell; Cell sorting; Flow cytometry; Mesenchymal stem cells; Pericyte; Tunica adventitia