Clin Orthop Surg.  2017 Jun;9(2):239-248. 10.4055/cios.2017.9.2.239.

Platelet-Derived Growth Factor Receptor-Positive Pericytic Cells of White Adipose Tissue from Critical Limb Ischemia Patients Display Mesenchymal Stem Cell-Like Properties

• Kim EJ
• Seo SG
• Shin HS
• Lee DJ
• Kim JH
• Lee DY

• KMID: 2412295
• DOI: http://doi.org/10.4055/cios.2017.9.2.239

Abstract

BACKGROUND
The pericytes in the blood vessel wall have recently been identified to be important in regulating vascular formation, stabilization, remodeling, and function. We isolated and identified pericyte-like platelet-derived growth factor receptor beta-positive (PDGFRβ+) cells from the stromal vascular fraction (SVF) of adipose tissue from critical limb ischemia (CLI) patients and investigated their potential as a reliable source of stem cells for cell-based therapy.
METHODS
De-identified subcutaneous fat tissues were harvested after amputation in CLI patients. Freshly isolated SVF cells and culture-expanded adipose-derived stem cells (ADSCs) were quantified using flow cytometry. A matrigel tube formation assay and multi-lineage differentiation were performed to assess pericytic and mesenchymal stem cell (MSC)-like characteristics of PDGFRβ+ ADSCs.
RESULTS
PDGFRβ+ cells were located in the pericytic area of various sizes of blood vessels and coexpressed mesenchymal stem cell markers. PDGFRβ+ cells in freshly isolated SVF cells expressed a higher level of stem cell markers (CD34 and CXCR4) and mesenchymal markers (CD13, CD44, CD54, and CD90) than PDGFRβ- cells. In vitro expansion of PDGFRβ+ cells resulted in enrichment of the perivascular mesenchymal stem-like (PDGFRβ+/CD90+/CD45-/CD31-) cell fractions. The Matrigel tube formation assay revealed that PDGFRβ+ cells were located in the peritubular area.
CONCLUSIONS
PDGFRβ+ ADSCs cells demonstrated a good multilineage differentiation potential. Pericyte-like PDGFRβ+ cells from the SVF of adipose tissue from CLI patients had MSC-like characteristics and could be amplified by in vitro culture with preservation of their cell characteristics. We believe PDGFRβ+ cells in the SVF of adipose tissue can be used as a reliable source of stem cells even in CLI patients.

Keyword

Platelet-derived growth factor; Adipose tissue; Adult stem cells; Ischemia

MeSH Terms

Adipose Tissue, White/*cytology/metabolism
Amputation
Cells, Cultured
Humans
Ischemia/*metabolism
Mesenchymal Stromal Cells/cytology/*metabolism
Pericytes/cytology/*metabolism
Platelet-Derived Growth Factor/*metabolism
Receptors, Platelet-Derived Growth Factor/*metabolism
Platelet-Derived Growth Factor
Receptors, Platelet-Derived Growth Factor

Figure

• 

  Fig. 1 Immunofluorescence of endothelial and perivascular markers in fresh white adipose tissue of human subjects. (A) H&E stain shows abundant vessels in the fat tissue including a small artery (large square), an arteriole (medium square), and a capillary (small square). The small artery (B), arteriole (C), and capillary (D) stained with indicated markers showed that platelet-derived growth factor receptor beta (PDGFRβ; CD140b)-positive (+) cells were located in the pericytic area while CD31 staining was exclusively visualized at the luminal surface for the small blood vessel. Costaining with a mesenchymal marker (CD90) showed that pericytic PDGFRβ+ cells also expressed the mesenchymal marker in small arterioles (C and D), while CD90-positive cells and PDGFRβ+ cells were mutually exclusive in the larger artery (B).

• 

  Fig. 2 Fluorescence-activated cell sorter analysis of fresh stromal vascular fraction. Platelet-derived growth factor receptor beta-positive (PDGFRβ+) cells expressed a higher level of stem cell markers (CD34 and CXCR4) and mesenchymal markers (CD13, CD44, CD54, and CD90) than PDGFRβ– cells. Cell populations with a positive pericyte marker (α-smooth muscle actin [αSMA]) or endothelial markers (CD31 and CD144) were more abundant in PDGFRβ+ cells. VEGFR: vascular endothelial growth factor receptor, NG: neuron-glial antigen.

• 

  Fig. 3 Fluorescence-activated cell sorter analysis of culture-expanded adipose-derived stem cell from fresh stromal vascular fraction (SVF) of human adipose tissue. A population with endothelial (CD31 and CD144) and leukocytic (CD45) markers was substantially reduced. Cultured platelet-derived growth factor receptor beta-positive (PDGFRβ+) cells maintained the feature of PDGFRβ+ cells from fresh SVF except that they had a lower level of stem cell marker (CD34 and CXCR4)-positive cells. Cultured PDGFRβ+ cells expressed high mesenchymal markers (CD13, CD44, CD54, and CD90) and a pericyte marker (α-smooth muscle actin [αSMA]). However, they did not express endothelial (CD31 and CD144) nor leukocytic (CD45) marker. VEGFR: vascular endothelial growth factor receptor, NG: neuron-glial antigen.

• 

  Fig. 4 Matrigel tube formation of fluorescence-activated cell sorter-sorted platelet-derived growth factor receptor beta-positive (PDGFRβ+) cells. Human umbilical vein endothelial cells (HUVECs) and CD140b (+) cells were labeled with von Willebrand factor (vWF; green) and CD140b (red), respectively. Nuclei were labeled by DAPI stain (blue). (A) Tubular network formation was more abundant when PDGFRβ+ adipose-derived stem cells (ADSCs) were cocultured with HUVECs (c) than when HUVEC only (a) or ADSC only (b) were cultured. (B) When PDGFR beta-positive (PDGFRβ+) ADSCs were cocultured with HUVECs, they showed the pericytic location of PDGFRβ+ ADSCs (red) which adhered to HUVECs (green) when observed at higher magnification using a confocal microscope.

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  Fig. 5 Differentiation assay of fluorescence-activated cell sorter-sorted platelet-derived growth factor receptor beta-positive (PDGFRβ+) cells. (A) In osteogenic differentiation, which was assayed with alkaline phosphatase (ALP) staining, the PDGFRβ+ cells showed greater ALP staining. (B) In chondrogenic differentiation, which was verified by Safranin O staining, the PDGFRβ+ cells showed greater chondrogenic differentiation. (C) After adipogenic induction for 3 days, the cell morphology changed from long spindle-shape into a round or polygonal shape. One week later, small bubble-shaped oil red O-staining lipid droplets appeared in part of the cells. ADSC: adipose-derived stem cells.

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