J Wang1, J Li, N Deng, X Zhao, Y Liu, X Wang, H Zhang. 1. Qilu Hospital of Shandong University Jinan, China2Department of Orthopedics Qingdao Hiser Medical Center, Qingdao, China3Department of Joint Surgery Affiliated Hospital of Qingdao University Qingdao, China - orthopedist_qd@163.com.
Abstract
AIM: Objective of the study was to compare the liposome-mediated transfection efficiency of the recombinant plasmid pIRES2-EGFP-hBMP-2 into mesenchymal stem cells (MSCs) derived from human umbilical cord blood (UCB-MSCs) and bone marrow (BM-MSCs). METHODS: UCB-MSCs and BM-MSCs were isolated using density gradient centrifugation followed by adherent cultures. The plasmid (pIRES2-EGFP-hBMP-2) was transfected into MSCs by using X-treme GENE transfection kit. Successful transfection was determined by both the protein expression of EGFP observed by fluorescence microscopy and the mRNA expression of human bone morphogenetic protein-2 (hBMP-2) detected using RT-PCR. Cell surface marker and morphological changes were examined by immunohistochemistry (IHC) after two weeks of transfection. RESULTS: Both UCB-MSCs and BM-MSCs were isolated and expanded successfully for multiple passages and demonstrated slight growth and morphological differences. The recombinant plasmid (pIRES2-EGFP-hBMP-2) was successfully transfected into both UCB-MSCs and BM-MSCs with an efficiency of 27.7 ± 7.6% and 18.4 ± 5.9%, respectively. The mRNA expression of hBMP-2 correlated with increased staining for collagen type II in the transfected cells. CONCLUSION: Transfection of hBMP-2 in UCB-MSCs and BM-MSCs increases the expression of collagen type II. These results indicate that increased BMP-2 levels induce multipotent stem cell differentiation into chondrocytes.
AIM: Objective of the study was to compare the liposome-mediated transfection efficiency of the recombinant plasmid pIRES2-EGFP-hBMP-2 into mesenchymal stem cells (MSCs) derived from human umbilical cord blood (UCB-MSCs) and bone marrow (BM-MSCs). METHODS: UCB-MSCs and BM-MSCs were isolated using density gradient centrifugation followed by adherent cultures. The plasmid (pIRES2-EGFP-hBMP-2) was transfected into MSCs by using X-treme GENE transfection kit. Successful transfection was determined by both the protein expression of EGFP observed by fluorescence microscopy and the mRNA expression of humanbone morphogenetic protein-2 (hBMP-2) detected using RT-PCR. Cell surface marker and morphological changes were examined by immunohistochemistry (IHC) after two weeks of transfection. RESULTS: Both UCB-MSCs and BM-MSCs were isolated and expanded successfully for multiple passages and demonstrated slight growth and morphological differences. The recombinant plasmid (pIRES2-EGFP-hBMP-2) was successfully transfected into both UCB-MSCs and BM-MSCs with an efficiency of 27.7 ± 7.6% and 18.4 ± 5.9%, respectively. The mRNA expression of hBMP-2 correlated with increased staining for collagen type II in the transfected cells. CONCLUSION: Transfection of hBMP-2 in UCB-MSCs and BM-MSCs increases the expression of collagen type II. These results indicate that increased BMP-2 levels induce multipotent stem cell differentiation into chondrocytes.
Authors: Sofia Bougioukli; Osamu Sugiyama; William Pannell; Brandon Ortega; Matthew H Tan; Amy H Tang; Robert Yoho; Daniel A Oakes; Jay R Lieberman Journal: Hum Gene Ther Date: 2018-03-14 Impact factor: 5.695