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

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.

Proteoglycan-4 is an Essential Regulator of Synovial Macrophage Polarization and Inflammatory Macrophage Joint Infiltration

Background: Synovial macrophages (SMs) perform a multitude of functions that include clearance of cell debris and foreign bodies, tissue immune surveillance, and resolution of inflammation. At one end of the macrophage polarization spectrum is the inflammatory phenotype (M1), which secretes IL-1β, IL-6 and expresses iNOS. On the opposite end of the spectrum is the anti-inflammatory phenotype (M2) which is characterized by the secretion of IL-10 and TGF-β. PRG4 is an important regulator of synovial hyperplasia and fibrotic remodeling and the involvement of SM activation in PRG4’s homeostatic role is yet to be defined. Our objectives were to study PRG4’s importance to SM homeostasis, M1 and M2 polarization and joint infiltration of bone marrow-derived macrophages (BMDMs) and investigate the role of SMs in mediating synovial fibrosis in Prg4 gene-trap (Prg4GT/GT) murine knee joints.Methods: SM phenotyping in Prg4GT/GT and Prg4+/+ joints was performed using flow cytometry and the balance between CD86+/CD206- (M1) and CD86-/CD206+ (M2) SMs was studied as animals aged. Expression of iNOS and IL-6 in CD86+ SMs, arginase-1 in CD206+ SMs and the impact of Prg4 recombination on SM polarization and BMDM infiltration following a TLR2 agonist challenge were determined. Inflammatory SMs were depleted using liposomal clodronate and synovial membrane thickness and expression of fibrotic markers: α-SMA, PLOD2 and collagen type I (COL-I) were assessed using immunohistochemistry.Results: Total macrophages in Prg4GT/GT joints were higher than corresponding age-matched Prg4+/+ joints (p<0.0001) and the percentages of CD86+/CD206- and CD86+/CD206+ SMs increased in Prg4GT/GT joints as animals aged (p<0.0001), whereas the percentage of CD86-/CD206+ SMs decreased (p<0.001). CD86+ SMs expressed iNOS and IL-6 compared to CD86- SMs (p<0.0001) while CD206+ SMs also expressed arginase-1. Prg4 re-expression limited the accumulation of CD86+ SMs, increased CD86-/CD206+ SMs and attenuated BMDM recruitment (p<0.001). Liposomal clodronate reduced inflammatory SMs and in turn reduced synovial hyperplasia, α-SMA, PLOD2 and COL-I expression in the synovium (p<0.0001).Conclusions: SM accumulation in the joint and the balance between inflammatory and anti-inflammatory SM subsets are regulated by PRG4. In the absence of PRG4’s role, the synovium is populated with inflammatory macrophages that drive synovial fibrosis.

Acceleration of Bone-Tendon Interface Healing by Low-Intensity Pulsed Ultrasound Is Mediated by Macrophages

Objective Low-intensity pulsed ultrasound (LIPUS) has been proven to facilitate bone-tendon interface (BTI) healing and regulate some inflammatory cytokines. However, the role of macrophages, a key type of inflammatory cell, during treatment remains unknown. This study aimed to investigate the role of macrophages in the treatment of BTI injury with LIPUS in a rotator cuff tear animal model. Methods In this experimental and comparative study, a total of 160 C57BL/6 mature male mice that underwent supraspinatus tendon detachment and repair were randomly assigned to 4 groups: daily ultrasonic treatment and liposomal clodronate (LIPUS+LC), daily ultrasonic treatment and liposomes (LIPUS), daily mock sonication and liposomal clodronate (LC), and daily mock sonication and liposomes (control). LIPUS treatment was initiated immediately postoperatively and continued daily until the end of the experimental period. Results The failure load and stiffness of the supraspinatus tendon–humerus junction were significantly higher in the LIPUS group than in the other groups at postoperative weeks 2 and 4, whereas those in the LIPUS+LC and LC groups were lower than those in the control group at postoperative week 4. The LIPUS, LIPUS+LC, and LC groups exhibited significantly more fibrocartilage than the control group at 2 weeks. Only the LIPUS group had more fibrocartilage than the control group at 4 weeks. Micro–computed tomography results indicated that LIPUS treatment could improve the bone quality of the attachment site after both 2 and 4 weeks. When macrophages were depleted by LC, the bone quality–promoting effect of LIPUS treatment was significantly reduced. Conclusions The enhancement of BTI healing by LIPUS might be mediated by macrophages. Impact In our study, LIPUS treatment appeared to accelerate BTI healing, which was associated with macrophages based on our murine rotator cuff repair model. The expressions of macrophage under LIPUS treatment may offer a potential mechanism to explain BTI healing and the effects of LIPUS on BTI healing.

873 Pharmacologic macrophage depletion affects metastasis formation by modulating systemic immune responses in a genetic pancreatic cancer model

BackgroundTumour-associated macrophages (TAM) play an important role in mediating tumour progression. In pancreatic cancer, infiltrating macrophages have been identified not only in invasive tumours, but also in early preinvasive pancreatic intraepithelial neoplasias and are known to mediate tumour progression.MethodsWe aimed to study the impact of pharmacological macrophage depletion by liposomal clodronate in the genetic mouse model of pancreatic cancer (KPC mouse: LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre). KPC mice were treated with liposomal clodronate or control liposomes from week 8 to week 20. Tumour and metastasis formation as well as alterations in local and circulating immune cells and cytokines were analysed.ResultsTreatment with liposomal clodronate effectively reduced CD11b-positive macrophages both in the pancreas and other organs such as liver, lung and spleen. Tumour incidence and size was only slightly reduced. However, metastasis formation in the liver und lungs was markedly diminished after macrophage depletion. Reduced macrophage count was associated with significant alterations in circulating growth factors and mediators known to be secreted by macrophages and associated with angiogenesis, most prominently VEGF. Moreover, application of liposomal clodronate led to marked alterations in circulating immune cells, among them reduced regulatory T cells.ConclusionsPharmacological depletion of macrophages in a genetic mouse model of pancreatic cancer markedly reduced metastasis formation and is associated with modulated profile of both secreted mediators and regulatory T cells. Pharmacological modulation of infiltrating macrophages represents a promising avenue for antimetastatic therapeutic approaches.