Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Sep 26, 2015; 7(8): 1137-1144
Published online Sep 26, 2015. doi: 10.4252/wjsc.v7.i8.1137
Easily-handled method to isolate mesenchymal stem cells from coagulated human bone marrow samples
Heng-Xiang Wang, Zhi-Yong Li, Zhi-Kun Guo, Zi-Kuan Guo
Heng-Xiang Wang, Department of Hematology, General Hospital of Air Force, Beijing 100042, China
Zhi-Yong Li, Zhi-Kun Guo, Key and Open Lab for Tissue Regeneration, Xinxiang Medical University, Xinxiang 453003, Henan Province, China
Zi-Kuan Guo, Department of Experimental Hematology, Beijing Radiation Medicine, Beijing 100850, China
Author contributions: Wang HX participated in the conception and designed the research; Li ZY and Guo ZhK performed the experiments and drafted the manuscript; Guo ZK deigned the study, analyzed the data, and prepared the manuscript; all authors had read and approved the final manuscript.
Supported by Natural Science Foundation of China, Nos. 30971068 and 81170522.
Institutional review board statement: This study was approved by General Hospital of Air Forces Review Board. The approval form has been uploaded via the submission website.
Conflict-of-interest statement: The authors have no conflicts of interest to disclose.
Data sharing statement: The letter of data sharing statement has been up-loaded via the submission website.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Zi-Kuan Guo, MD, PhD, Department of Experimental Hematology, Beijing Radiation Medicine, 27# Taiping Road, Beijing 100850, China. guozk@bmi.ac.cn
Telephone: +86-10-68158312 Fax: +86-10-68158312
Received: April 10, 2015
Peer-review started: April 12, 2015
First decision: May 14, 2015
Revised: June 30, 2015
Accepted: August 4, 2015
Article in press: August 7, 2015
Published online: September 26, 2015
Processing time: 168 Days and 6.5 Hours
Abstract

AIM: To establish an easily-handled method to isolate mesenchymal stem cells (MSCs) from coagulated human bone marrow samples.

METHODS: Thrombin was added to aliquots of seven heparinized human bone marrow samples to mimic marrow coagulation. The clots were untreated, treated with urokinase or mechanically cut into pieces before culture for MSCs. The un-coagulated samples and the clots were also stored at 4 °C for 8 or 16 h before the treatment. The numbers of colony-forming unit-fibroblast (CFU-F) in the different samples were determined. The adherent cells from different groups were passaged and their surface profile was analyzed with flow cytometry. Their capacities of in vitro osteogenesis and adipogenesis were observed after the cells were exposed to specific inductive agents.

RESULTS: The average CFU-F number of urokinase-treated samples (16.85 ± 11.77/106) was comparable to that of un-coagulated control samples (20.22 ± 10.65/106, P = 0.293), which was significantly higher than those of mechanically-cut clots (6.5 ± 5.32/106, P < 0.01) and untreated clots (1.95 ± 1.86/106, P < 0.01). The CFU-F numbers decreased after samples were stored, but those of control and urokinase-treated clots remained higher than the other two groups. Consistently, the numbers of the attached cells at passage 0 were higher in control and urokinase-treated clots than those of mechanically-cut clots and untreated clots. The attached cells were fibroblast-like in morphology and homogenously positive for CD44, CD73 and CD90, and negative for CD31 and CD45. Also, they could be induced to differentiate into osteoblasts and adipocytes in vitro.

CONCLUSION: Urokinase pretreatment is an optimal strategy to isolate MSCs from human bone marrow samples that are poorly aspirated and clotted.

Keywords: Coagulated marrow sample; Mesenchymal stem cells; Isolation; Urokinase

Core tip: It is usually difficult to isolate mesenchymal stem cells (MSCs) from bone marrow samples that have been coagulated due to inappropriate marrow aspirations or in the setting of pathologically hypercoagulative state. In this study, the authors found that urokinase pretreatment could dissolve stem cells from the clots. The MSCs were fibroblat-like in morphology and homogenously positive for CD44, CD73 and CD90, and negative for CD31 and CD45. Also, they could be induced to differentiate into osteoblasts and adipocytes in vitro. Urokinase pretreatment is an optimal strategy to isolate MSCs from poorly aspirated and clotted human bone marrow samples.