Modified method for effective primary vascular smooth muscle progenitor cell culture from peripheral blood

Jin-Hee Seong,A-Hyeon Lee,Amy M Kwon,Na-Kyoung Shin,Kyung-Soo Kim,Guang-Yin Shen,In-Hwa Park,Yi-Sun Song,Yonggu Lee,Hyun-Woo Joo,Hyuck Kim

doi:10.1007/s10616-020-00419-2

Abstract

In previous studies, vascular smooth muscle progenitor cells (vSMPCs) isolated from peripheral blood mononuclear cells (PBMCs) were cultured using medium containing platelet-derived growth factor-BB (PDGF-BB) for 4 weeks. However, this method requires long culture periods of up to 4 weeks and yields low cell counts. Therefore, we proposed the modified method to improve the cell yield and purity and to reduce the cell culture period. PBMCs were isolated from human peripheral blood and cultured by the conventional method using medium containing PDGF-BB alone or the modified method using medium containing PDGF-BB, basic fibroblast growth factor (bFGF), and insulin-transferrin-selenium ITS for 4 weeks. The purity of vSMPCs was analyzed for the expression of a- smooth muscle actin (SMA) by flow cytometry and significantly higher in the modified method than conventional methods at the 1st and 2nd weeks. Also, mRNA expression of a-SMA by real-time PCR was significantly higher in the modified method than conventional method at the 2 weeks. The yield of vSMPCs by trypan blue exclusion assay was significantly higher in the modified method than conventional method at the 1st, 2nd and 3rd weeks. The primary culture using the modified method with PDGF-BB, bFGF, and ITS not only improved cell purity and yield, but also shortened the culture period, compared to the conventional culture method for vSMPCs. The modified method will be a time-saving and useful tool in various studies related to vascular pathology.

Highlights

Vascular smooth muscle progenitor cells are defined as circulating or resident cells localized within the bone marrow (BM), vascular wall, or perivascular area that are committed to the smooth muscle fate and capable of differentiating into mature smooth muscle cells (SMCs) (Merkulova-Rainon et al 2012)
After washing 3 times with phosphate buffered saline (PBS; Gibco, Grand Island, NY, U.S.), the peripheral blood mononuclear cells (PBMCs) was cultured in dulbecco’s modified eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) containing 20% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA); 1% 100 U/ml penicillin and 100 mg/ml streptomycin (P.S; Gibco, Grand Island, NY, USA); 50 ng/ml platelet-derived growth factor-BB (PDGF-BB) (R&D systems Inc., Minneapolis, Minnesota, U.S.) (Conventional method) or DMEM containing 20% FBS; 1% P.S; 5 ng/ml PDGF-BB; 10 ng/ml basic fibroblast growth factor (bFGF) (Biobud, Seongnam-si, Gyeonggi-do, Republic of Korea); ITS (10 ng/ml insulin-5.5 ng/ml Transferrin–5 ng/ml Selenium; Gibco, Grand Island, NY, U.S.) (Modified method) and placed on 6 well plate coated with type I collagen (Corning, Corning, New York, U.S.) at a density of 2 9 106/well
Purity of vascular smooth muscle progenitor cells according to culture period In order to compare the purity of vSMPCs according to culture period, the percentage of a-smooth muscle actin (SMA) positive cells was analyzed by Fluorescence-activated cell sorting (FACS) (Fig. 2 and Table 1)

Summary

Introduction

Vascular smooth muscle progenitor cells (vSMPCs) are defined as circulating or resident cells localized within the bone marrow (BM), vascular wall, or perivascular area that are committed to the smooth muscle fate and capable of differentiating into mature smooth muscle cells (SMCs) (Merkulova-Rainon et al 2012). Previous reports have described that vSMPCs could be cultured from peripheral blood mononuclear cells (PBMCs) in the medium containing PDGF-BB (Kang et al 2014; Lin et al 2016; Simper et al 2002; Sugiyama et al 2006; Xie et al 2008). This conventional primary culture method requires at least a 4-week-long culture period and generally yields low cells counts

Methods

Results

Conclusion