1 College of Basic Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China National Key Laboratory of Collateral Disease Research and Innovative Chinese Medicine, Shijiazhuang 050035, China.Abbreviations: ACL, anterior cruciate ligament APC, allophycocyanin Cy7, cyanine 7 FITC, fluorescein isothiocyanate PE, phycoerythrin SSC‐A, side scatter‐area SSC‐H, side scatter‐height TKR, total knee replacement. (D): The percentage of the live cells from the stromal vascular fraction that were pericytes or adventitial cells were compared. (C): The number of cells sorted per gram of tissue were compared between patients undergoing ACL reconstruction or TKR. (B): Mean number of pericytes, adventitial cells, and perivascular stem cells sorted using fluorescence‐activated cell sorting from patients undergoing ACL reconstruction or TKR were compared. Gates used to select pericytes (CD146+CD34−) and adventitial cells (CD146−CD34+ bottom middle) were based on isotype controls (bottom right). Endothelial cells and hematopoietic stem cells were excluded based on expression of CD31 and CD45, respectively (bottom left). Events likely to represent single cells were selected on their size and relative dimensions, followed by dead cell exclusion using 4′,6‐diamidino‐2‐phenylindole (DAPI top row). (A): Fluorescence‐activated cell sorting images demonstrate the gating strategy used to isolate perivascular stem cells. on behalf of AlphaMed Press.įlow cytometry isolation of perivascular stem cells. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. Stem Cells Translational Medicine 2017 6:77-87.Īdipose Adult stem cells CD34+ Chondrogenesis Pericytes. PSCs generated significantly more extracellular matrix than culture-derived MSCs. The IFP was a significantly better source of chondrogenic stem cells compared with bone marrow. Micromass culture demonstrated that differentiated PSCs were upregulated compared with MSCs for COL2A1, ACAN, and SOX9 expression by factors of 4.8 ± 1.3, 4.3 ± 0.9, and 7.0 ± 1.7, respectively. The IFP PSCs generated significantly more extracellular matrix than IFP MSCs (p =. Using a pellet model, the IFP PSCs and the MSCs generated significantly more extracellular matrix than bone marrow MSCs (p <. Differentiation was confirmed using histochemical stains and genetic expression. Fluorescence-activated cell sorting demonstrated that cultured PSCs were CD44+CD90+CD105+ polymerase chain reaction and immunocytochemistry demonstrated that pericytes retained their CD146+ phenotype and expressed the pericyte markers PDGFRβ and NG2. The mean numbers of pericytes and adventitial cells isolated were 4.6 ± 2.2 × 10 4 and 16.2 ± 3.2 × 10 4, respectively, equating to 7.9 ± 4.4 × 10 3 and 20.8 ± 4.3 × 10 3 cells per gram of harvested tissue. Pericytes and adventitial cells were isolated from the stromal vascular fraction (3.8% and 21.2%, respectively) using flow cytometry with a viability of 88%. Immunohistochemistry demonstrated the location of perivascular markers (CD146, CD34, neural/glial antigen 2, platelet-derived growth factor receptor-β, and α-smooth muscle actin ) in relation to endothelial markers (CD31, CD144, von Willebrand factor ). This research assessed the chondrogenic potential of IFP PSCs compared with MSCs from the IFP and bone marrow. Cells from the infrapatellar fat pad (IFP) have demonstrated increased chondrogenic potential compared with those from subcutaneous fat. Prospectively identified and isolated PSCs have demonstrated increased plasticity and osteogenic potential. Perivascular stem cells (PSCs) are the natural ancestors of mesenchymal stem cells (MSCs) and are the stem cells responsible for homeostasis and repair in vivo.
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