| BACKGROUNDHematopoietic stem cell transplantation (HSCT) is a procedure most often performed for patients with hematologic malignancies, solid tumors and patients with certain genetic diseases. In these cases the recipient's hematopoietic and immune system is destroyed by high-dose radiotherapy and chemotherapy before transplantation which maybe autologous or allogeneic. Currently, HSCT is the only treatment of hematologic malignancies, such as leukemia and lymphoma, etc. Infections and GVHD are major complications of allogenic HSCT. After HSCT, the vast majority of transplant complications and deaths occur before the hematopoietic and immune reconstitution. Fast and stable hematopoietic reconstitution is the most critical step of immune reconstitution. Therefore, rapid and effective reconstruction can significantly affect the survival rate and quality of life after HSCT.Cell therapy is at present mostly infused by intravenous route, the cells are distributed throughout the whole body before reaching the target organs and tissues, for instance in HSCT, only (1.56±0.32)% hematopoietic stem cells home to the bone marrow at the first 20 h after transplantation, the vast majority of transplanted cells were arrested in the pulmonary circulation, liver and spleen by conventional intravenous infusion. Obviously, hematopoietic stem cells cannot re-establish hematopoiesis unless they have homed back and engrfted into bone marrow. Efforts have been undertaken by researchers to direct MSCs to the target tissue by co-infusion with hematopoietic stem cells, however low efficiency of MSCs transplantation remains a challenge. Bone and bone marrow is composed of specific anatomical structure and physiological environment. It is difficult for the injection cells to migrate, gather and grow in the "niche" effectively just relying on the various factors, receptors, regulation and chemotaxis existing in the bone marrow microenvironment.Is it possible for the stem cell target transplantation in bone marrow? How is cell target migration achieved efficiently? As our study entitled, Magnetism-Induced Cell Target Transplantation (MagiC-TT) could be a solution to this problem. The concept of Magic-TT is that donor cells are magnetized firstly then are induced to target transplant to recipients'specific organ, such as bone marrow. Under the help of magnetic field, donor cells are homed back to the bone marrow rapidly, locally distribute and get implanted in the niche.The most important cells during the process of HSCT include hematopoietic stem/progenitor cells (HS/PCs) and mesenchymal stem cells (MSCs) which participate in inter-forming hematopoietic microenvironment. HS/PCs is the first kind of adult stem cells carried out in the area of stem cell research and clinical application, carrying an extremely important value in HSCT, gene therapy, immunotherapy and preparation of blood products in vitro. MSC is a mature, fibroblast-like pluripotent cells derived from the development of early mesoderm, capable of differentiating into fat cells, nerve cells, heart cells, chondrocytes and osteoblasts[14-16]. MSC can also migrate to the target organs or tissue, however not very efficiently, to secrete large amounts of immune regulation, anti-inflammatory, anti-apoptotic, angiogenic, chemokines and other bioactive molecules, so it is widely used in HSCT-related diseases. Due to these characterizing features, HS/PCs and MSCs are used in the study of MagiC-TT to achieve stem cells target transplantation.OBJECTIVE1 Ex vivo experiment:Magnetization of red fluorescent protein MSCs (RFP-MSCs) with specific-made Ferric Oxide Nanoparticles (Fe3O4@PDA@Au NPs) and the study of magnetization effect of RFP-MSCs.2 In vivo experiment of exploration of the effect of bone marrow target transplantation and the distribution and survival of magnetized RFP-MSCs mediated by MagiC-TT.3 Autologous HSCT mouse model:Study of hematopoietic reconstitution of irradiated recipient mice after HS/PCs transplantation by MagIC-TT.CONTENTSPart 1. Ex vivo magnetization of RFP-MSCs with Fe3O4@PDA@Au NPs and the study of magnetization effect of RFP-MSCs induced by MagiC-TT.[Methods] (1) To introduce Fe3O4@PDA@Au NPs into RFP-MSCs, sort out magnetized cells and calculate the positive rate of magnetization. (2) Magnetized and wild type RFP-MSCs were compared based on cell morphology, proliferation, differentiation and immune phenotype to comprehend the biological safety and compatibility of Fe3O4@PDA@Au NPs. (3) The distribution of NPs in magnetized RFP-MSCs, and their ability of magnetized RFP-MSCs to migrate to target tissues and engraftment under magnetic field. (4) The magnetized and wild type NeoR-Luc-RFP-MSCs mixed with luciferase substrate were injected into the dissociated murine femurs, then the migration of cells was observed by using the bioluminescent imaging system.[Res?lts] (1) The positive rate of Fe3O4@PDA@Au NPs magnetization was (85.36±1.24)%. (2) There was no variation in basic characteristics of magnetized and wild type RFP-MSCs. NPs exist within or on the surface of RFP-MSCs. (3) The magnetized RFP-MSCs were capable of target migration under magnetic field. (4) It was observed that the site of the fluorescence changed with the movement of the magnetism by bioluminescent imaging system.[Conclusions]This part of the experiment proved that Fe3O4@PDA@Au NPs have good biocompatibility and are non-toxic. Ex vivo MSCs can be magnetized by a new type of magnetic composites, Fe3O4@PDA@Au NPs, thus rendering them capable of target transplantation by MagiC-TT technique.Part 2. In vivo target transplantation of magnetized RFP-MSCs induced by Fe3O4@PDA@Au NPs.[Methods] (1) NeoR-Luc-RFP-MSCs experiment:Eight wild-type BALB/C female mice were evenly divided randomly into experimental group (M group) and control group (C group). Magnetized NeoR-Luc-RFP-MSCs were injected into bone marrow of mice in both groups, with or without magnetism on femur (M or C group). Fluorescent changes in both groups of mice at different time points were observed by bioluminescence. (2) RFP-MSCs experiment:20 green fluorescent protein (GFP) transgenic C57BL mice were randomly divided into M group (n=10) and C group (n=10). Magnetized RFP-MSCs were transplanted into the femur cavity of the mice with the help of X-ray, under magnetic field in the M group and without magnetic field in the C group. Bioluminescence, FACS analysis, PCR, histopathological analysis and self-developed semi-solid decalcification (SSD) technology were performed to study the biological safety and targeted migration effect of RFP-MSCs post-transplantation.[Res?lts] (1) Bioluminescence assay showed that RFP-MSCs appeared in the lung of C group mice 5 min after cell injection while the magnetized RFP-MSCs were limited to the femur of M group mice. However, on withdrawal of the magnetic field 1h later, strong fluorescence was observed in the lung of M group within 5 min and the fluorescence peaked at 15 min. (2) By pathological examination, flow cytometry and PCR, a large number of RFP-MSCs were observed to reside within the bone marrow in M group while few in C group. (3) SSD system of pathological analysis clearly showed the location, shape and interaction of red/green fluorescent cells (donor/recipient cells) after transplantation. (4) RFP-MSCs were found to survive more than 3 months in different organs in both groups.[Conclusions] This part of the experiment proved that RFP-MSCs mainly distributed in the lungs, liver, spleen and other organs in the Group C mice; while magnetized RFP-MSCs can be successfully migrated to target site by using MagiC-TT method in vivo. After the magnetic field was removed 1 h after, the magnetized cells could migrate in the body. Magnetized cells in mice were non-toxic and could survive for a long time in vivo.Part 3. The study of hematopoietic reconstitution after HS/PCs transplantation by MagiC-TT in the mouse model of autologous HSCT.[Methods] (1) Number gradient experiment of transplant cells:60 C57BL/6 mice were randomly divided into 6 groups (n=10). HS/PCs were injected via the femur (three gradients of 5×106?1×106, 1×105) and the tail vein (three gradients of 5×106? 1×106, 1×105), respectively. After myeloablative radiotherapy by 7.5Gy linear accelerator, cells were injected, the survival rate of mice was observed in each group. (2) HS/PCs experiment of femur target transplantation:34 C57BL/6 mice were randomly divided into two groups (n=17). After 7.5Gy myeloablative radiotherapy, both groups of mice were injected with 0.02ml PBS of cells suspension through femur (1×106). Right femur was induced with magnetic field (M group) or without magnetic field (C group). FCM was used to detect the number and proportion of GFP+ cells in right and left femur of both groups of mice at different time points (Oh, 24h,72h) after cells injection. The survival, blood cell count and GFP+ cells in peripheral blood were observed in the remaining mice (8 in each group). The distribution of GFP+ cells in the body was explored by using pathological examination, SSD, FCM and Q-PCR.(Res?lts] (1) Number gradient of transplanted cells in experimental group:the survival of 6 out of 10 mice in tail vein infusion of 5x106 cells, while none of mice infused with 1×105 and 1×106cells survive, respectively. Femoral injection of 5×106 cells lead to survival of all the mice in the group,2 out of 10 mice survived in the group infused with 1×106 cells, however those infused with 1×105 cells all died. (2) HS/PCs femur target transplantation:a. Mice in W group and C group lost weight and showed reduced activity after radiotherapy; mental condition and physical movement began to recover 1 week later, b.7 out of 8 mice survived more than 30d in M group while 1 out of 8 mice remained alive within 30d in C group. Survival time of mice between the two groups has significant statistically difference (P<0.05). c. During hematopoietic recovery, the proportions of GFP+cells in peripheral blood of mice in M group and C group were (94.10±1.97)% and 93.82%, respectively, d.0 h,24 h,72 h after cells infusion, FCM results of GFP+cells in the femur of both groups of mice showed that the number of cells in right femur were more than that of the left femur. Significant statistical difference in M group recorded as (Poh=0.040, P24h=0.030, P72h=0.049) but no statistical difference was seen in C group (Poh=0.184, P24h=0.184, P72h=0.368). The number of cells in right (cell injected) femur of the mice in M group was more than that in C group; statistical difference recorded as (P0h=0.007,P24h=0.006, P72h=0.036) but no statistical difference was observed in left (control) femur (P0h= 1.000, P24h=0.083,P72h=0.108). e. Platelet recovery in M group is faster than that of C group (12.33±2.42)d vs. (16.38±2.39)d, P=0.009, the lowest value of hemoglobin in M group was higher than that of C group (43.75±13.02)g/L vs. (13.75±5.18)g/L, P<0.001. f. Pathological treatment by SSD technology, clearly defined the exact position, shape and interaction of donor and recipient cells after HSCT.[Conclusions] This part of the experiment using the model of eGFP mice is auto-HSCT to explore the impact of mouse hematopoietic recovery after transplantation by MagiC-TT method. The results showed that magnetized HS/PCs in the auto-HSCT of GFP mouse achieved femur target transplantation by MagiC-TT, reducing mouse hematopoietic recovery time after transplantation and increasing the survival rate.SUMMARYOur study of MagiC-TT successf?lly acquired target transplantation based on the excellent characteristics of a new type of magnetic composites-Fe3O4@PDA @Au NPs. In combination with such technologies as SSD, MagiC-TT can be used for the study of cells homing, proliferation and mechanism of cell interaction. Hence, MagiC-TT creates a solid foundation for the future applications in research and clinics. |
PDF Full Text Request