Abstract
BACKGROUND:The use of mouse bone marrow mesenchymal stem cells (mBMSCs) represents a promising strategy for performing preclinical studies in the field of cell-based regenerative medicine; however, mBMSCs obtained via conventional isolation methods have two drawbacks, i.e., (i) they are heterogeneous due to frequent macrophage contamination, and (ii) they require long-term culturing for expansion.METHODS:In the present study, we report a novel strategy to generate highly pure mBMSCs using liposomal clodronate. This approach is based on the properties of the two cell populations, i.e., BMSCs (to adhere to the plasticware in culture dishes) and macrophages (to phagocytose liposomes).RESULTS:Liposomal clodronate added during the first passage of whole bone marrow culture was selectively engulfed by macrophages in the heterogeneous cell population, resulting in their effective elimination without affecting the MSCs. This method allowed the generation of numerous high-purity Sca-1+CD44+F4/80− mBMSCs (> 95%) with just one passaging. Comparative studies with mBMSCs obtained using conventional methods revealed that the mBMSCs obtained in the present study had remarkably improved experimental utilities, as demonstrated by in vitro multilineage differentiation and in vivo ectopic bone formation assays.CONCLUSION:Our newly developed method, which enables the isolation of mBMSCs using simple and convenient protocol, will aid preclinical studies based on the use of MSCs.
Highlights
Mesenchymal stem cells (MSCs) maintain undifferentiated state through their self-renewal and can further differentiate into various cells types such as osteoblasts, adipocytes, chondrocytes, and neurons under certain conditions [1]
We tried to solve the problems associated with the conventional mBMSC separation method by focusing on reducing the contamination with non-MSCs
We confirmed that repeated passaging by controlled trypsinization of the BMSC culture, starting from the Bone marrow (BM) cells gradually reduces the number of macrophages and increases the percentage of MSCs (Fig. 1)
Summary
Mesenchymal stem cells (MSCs) maintain undifferentiated state through their self-renewal and can further differentiate into various cells types such as osteoblasts, adipocytes, chondrocytes, and neurons under certain conditions [1] Owing to their pluripotent properties in combination with their accessibility and low immunogenicity, MSCs are considered as potential candidates for developing cell-based therapy for damaged tissues and degenerative diseases [2, 3]. In order to solve the issues related to plastic adherence, unique purification methods, such as sorting by using antibodies against specific markers expressed on the cell surface or culture in hypoxic conditions, have been developed by several research groups [12,13,14,15] These techniques allow the selection of MSCs with improved functional clonogenicity, they may raise ethical issues as more mice would be sacrificed to obtain a limited yield of MSCs, a very inefficient use of resources. In the present study, we describe a simple and effective method for isolating pure MSCs from the mouse BM using liposomal clodronate
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