Original Article
Copyright ©2011 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Stem Cells. May 26, 2011; 3(5): 43-52
Published online May 26, 2011. doi: 10.4252/wjsc.v3.i5.43
Preclinical transplantation and safety of HS/PCs expanded from human umbilical cord blood
Chun-Juan Guo, Ying Gao, Di Hou, Dong-Yan Shi, Xiang-Min Tong, Dan Shen, Yong-Mei Xi, Jin-Fu Wang
Chun-Juan Guo, Di Hou, Dong-Yan Shi, Yong-Mei Xi, Jin-Fu Wang, College of Life Sciences, Zhejiang University, Hangzhou 310058, Zhejiang Province, China
Ying Gao, The Second People’s Hospital of Hangzhou, Hangzhou 310015, Zhejiang Province, China
Xiang-Min Tong, Dan Shen, The First Auxiliary Hospital of Zhejiang University, Hangzhou 310003, Zhejiang Province, China
Author contributions: Guo CJ participated in the experimental design, HS/PC expansion and analysis, determination of contamination in HS/PC product, interpretation of the data and was the primary author of the manuscript; Gao Y collected human umbilical cord blood and isolated hematopoietic stem cells, and performed the engraftment assay of HS/PC products in NOD/SCID mice; Hou D conceived the experiment, performed the culture of BM-MSCs; Shi DY detected the bacterial endotoxin concentration and stem cell factor; Tong XM collected human BM; Shen D isolated MSCs from BM; Xi YM participated in manuscript preparation; Wang JF supervised the study design, execution, analysis, and approved the final version.
Supported by Scientific Foundation of Zhejiang (2009C13020) and National Natural Science Foundation of China, No. 30971460
Correspondence to: Jin-Fu Wang, Professor, Institute of Cell Biology and Genetics, College of Life Sciences, Zi Jin Gang Campus, Zhejiang University, Hangzhou 310058, Zhejiang Province, China. wjfu@zju.edu.cn
Telephone: +86-571-88206592 Fax: +86-571-85128776
Received: August 10, 2010
Revised: January 5, 2011
Accepted: January 12, 2011
Published online: May 26, 2011
Abstract

AIM: To expand hematopoietic/progenitor stem cells (HS/PCs) from umbilical cord blood (UCB) and prepare the HS/PC product, and analyze preclinical transplantation and safety of HS/PC product.

METHODS: Human bone marrow-derived mesenchymal stem cells (MSCs) were used as feeder cells to expand HS/PCs from UCB in a serum-free culture system. The proliferation potential of HS/PCs was analyzed. The expanded HS/PCs were suspended in the L-15 medium to prepare the HS/PC product. The contamination of bacteria, fungi and mycoplasmas, the infection of exogenous virus, the concentration of bacterial endotoxin, and the SCF residual in HS/PC product were determined. Finally, cells from the HS/PC product with or without bone marrow-derived mesenchymal stem cells (BM-MSCs) were transplanted into the irradiated NOD/SCID mice to determine the in vivo engraftment potential.

RESULTS: After co-culture for 10 d, the total nuclear cells (TNCs) increased 125-fold, and CD34+ cells increased 43-fold. The granulocyte-macrophage colony- forming cells (GM-CFCs) and erythroid colony-forming cells (E-CFCs) increased 3.3- and 4.7-fold respectively. The expanded cells were collected and prepared as the expanded product of HS/PCs by re-suspending cells in L-15 medium. For preclinical safety, the HS/PC product was analysed for contamination by bacteria, fungi and mycoplasmas, the bacterial endotoxin concentration and the SCF content. The results showed that the HS/PC product contained no bacteria, fungi or mycoplasmas. The bacterial endotoxin concentration was less than the detection limit of 6 EU/mL, and residual SCF was 75 pg/mL. Based on clinical safety, the HS/PC product was qualified for clinical transplantation. Finally, the HS/PC product was transplanted the irradiated mice where it resulted in rapid engraftment of hematopoietic cells.

CONCLUSION: HSPC product prepared from UCB in the serum-free culture system with hMSCs as feeder cells should be clinically safe and effective for clinical transplantation.

Keywords: Hematopoietic stem cells, Ex vivo expansion, Preclinical safeties, Transplantation