Isolation of High Purity Mouse Mesenchymal Stem Cells through Depleting Macrophages Using Liposomal Clodronate

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.

Clodronate liposomes may biases MSC differentiation toward adipogenesis through activation of NLRP3

“…Our study found that the adipocytes in the bone marrow cavity were significantly increased by Clod-Lipo injection after HSCT, and we suspected that the increase of adipocytes in the bone marrow cavity of mice was related to MSC. The addition of Clod to primary MSC culture results in selective phagocytosis by macrophages in heterogeneous cell populations, resulting in effective removal of macrophages without affecting MSC and increasing MSC purity [ 29 ]. By culturing primary MSC in vitro followed by passaging, the fourth generation cells were identified as MSC by surface markers, and lipogenesis assay was performed after Clod-Lipo action, and the increased formation of lipid droplets was found by Oil Red O staining, which indicated that Clod-Lipo would induce MSC to bias towards adipogenesis.…”

Norisoboldine, a Natural Alkaloid from Lindera aggregata (Sims) Kosterm, Promotes Osteogenic Differentiation via S6K1 Signaling Pathway and Prevents Bone Loss in OVX Mice

“…[ 42 ] As previously reported methods, BMSCs were isolated from 8‐ to 12‐week‐old WT mice. [ 43 ] Briefly, the bone marrow of the femur and tibia was flushed out with PBS (P1020, Solarbio, Beijing, China). Osteoblasts and BMSCs were incubated in the basic medium (α‐MEM [10‐022CV, Corning, NY, USA] containing 10% FBS [35‐081‐CV, Corning, NY, USA] and 1% penicillin–streptomycin solution [C0222, Beyotime, Shanghai, China]).…”

Norisoboldine, a Natural Isoquinoline Alkaloid, Inhibits Diaphyseal Fracture Healing in Mice by Alleviating Cartilage Formation

“…BMSCs were isolated from 8-to 12-week-old WT mice, as previously reported [15]. Briefly, the bone marrow of the femur and tibia was flushed out with PBS and incubated in a basic medium [α-MEM (Corning, NY, USA) containing 10% FBS (Corning, USA) and 1% penicillin/streptomycin].…”