TY - JOUR
T1 - Prospective purification of perivascular presumptive mesenchymal stem cells from human adipose tissue
T2 - Process optimization and cell population metrics across a large cohort of diverse demographics
AU - West, C. C.
AU - Hardy, W. R.
AU - Murray, I. R.
AU - James, A. W.
AU - Corselli, M.
AU - Pang, S.
AU - Black, C.
AU - Lobo, S. E.
AU - Sukhija, K.
AU - Liang, P.
AU - Lagishetty, V.
AU - Hay, D. C.
AU - March, K. L.
AU - Ting, K.
AU - Soo, C.
AU - Péault, B.
N1 - Funding Information:
The authors would like to acknowledge the support they have received from the British Heart Foundation (Research Grant # R42775), the Medical Research Council, and the California Institute for Regenerative Medicine. CCW was supported by a Chief Scientist Office Clinical Academic Training Fellowship, a research grant from the William Rooney Plastic Surgery and Reconstructive Surgery Trust, and a Research Fellowship from the Royal College of Surgeons of England. KLM and WRH received support from a VA Merit Review grant, VA Center for Regenerative Medicine, NIH CCTRN grant UM1 5UM1HL113457-04, and NIH T32 grant 5T32HL079995-10. IRM was supported by a Wellcome Trust funded Edinburgh Clinical Academic Track (ECAT) Lectureship (ref. 097483).
Publisher Copyright:
© 2016 West et al.
PY - 2016/3/30
Y1 - 2016/3/30
N2 - 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.
AB - 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.
KW - Adipose tissue
KW - Adipose-derived stem cell
KW - Cell sorting
KW - Flow cytometry
KW - Mesenchymal stem cells
KW - Pericyte
KW - Tunica adventitia
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U2 - 10.1186/s13287-016-0302-7
DO - 10.1186/s13287-016-0302-7
M3 - Article
C2 - 27029948
AN - SCOPUS:84966263477
SN - 1757-6512
VL - 7
JO - Stem Cell Research and Therapy
JF - Stem Cell Research and Therapy
IS - 1
M1 - 47
ER -