Search

Background: Hematopoietic stem cells (HSCs) are maintained in a particular microenvironment termed as "niche", which are constructed by a number of critical molecules, supporting matrix such as collagen and supporting cells like osteoblasts and bone mesenchymal stem cells (BMSCs), together with physical construct of trabecular bone and endosteum. However, the mechanism of niche is poorly understood since it is surrounded by hard and opaque cortex of bone. Here, we developed a three-dimensional (3D) culture model using the deproteined and degreased human bone or ¦Â-TCP biomaterial, to mimic hematopoietic niche which is highly similar to nature situation and is easy to be observed. Objective: To establish the 3D culture model to mimic the hematopoietic niche with the deproteined and degreased human bone or ¦Â-TCP, where both have an architecture of trabecular bone; the HSC/progeny and supporting cells were gene-marked with GFP and RFP respectively; the process by which the HSCs differentiated to megakaryocyte/platelets were observed within this 3D culture model. Method: 1) 3D culture system: The human bone was obtained by bone biopsy from volunteer donor after the signing of Informed Consent£¬then was deproteined with 30%H2O2 for 2h and degreased with a mixture buffer of Chloroform, methanol and deionized water£¨Vc:Vm:Vw=9:9:2£© in 37¡æ for 24h. ¦Â-tricalcium phosphate (¦Â-TCP) biomaterial was bought from Bio-lu company (France), which is a kind of artificial, mass producible biomimetic macroporous material and clinically proven tissue-engineered bone. Both materials were cut into small pieces with 2-3mm in height and parallel tested. 2) Cells: The C57BL/6 RFP-BMSCs and its complete culture medium were bought from Cyagen Biosciences Inc. (China). Sca-1+ cells were harvested from the bone marrow of GFP transgenic mice by FACS sorting (GFP-sca-1+ cells). RFP-BMSCs and GFP-sca-1+ cells were inoculated into the 3D culture system. 100ng/mL thrombopoietin and 100 ng/mL platelet-derived growth factor (PDGF) were added into co-culture medium for 5 days in 3cm confocal microscopy dishes, both cells were also directly (2D) cultured as control. 3) Observation and Semi-solid decalcification (SSD, 2010 ASH poster, no.2625): The 3D culture system and controls were observed directly by a confocal laser scanning microscope (Olympus), 430nm for GFP and 559nm for RFP. The 3D culture system were also decalcified with self-made SSD and observed to clarify the interaction of GFP and RFP cells. Result: When GFP-sca-1+ cells were co-cultured with RFP-BMSCs for 12h, GFP+ cells attached to grow with RFP-BMSCs, two colors mixed with different morphology (Fig.1a). In the following days, with the stimulating of PDGF and TPO, most GFP cells, surrounded by RFP-BMSCs, were bigger and bigger with more and more antennae and floss (Fig.1b). Hematogenesis happens nearby the architecture of trabecular bone. With the help of SSD, the hard and opaque construction disappeared gradually, only GFP and RFP cells stayed where they were with their original morphology and actions. By frozen section, we can easily distinguish the cell type, developmental stage and interactions between each cell. Discussion: The standard 2D cell culture systems neither reflect the normal architecture, nor material properties such as stiffness, nor the diffusion of soluble factors of the natural system. The deproteined and degreased human bone we used, not only reproduced the spongy architecture of the niche, but also provide a scaffold for cell growth. Furthermore, the self-made SSD can ¡°digest¡± the scaffold but preserve the cells; it is convenient to observe the cells within the ¡°black box¡± in 3D. However, the human bone was hard to obtain. Therefore, we investigated it with ¦Â-TCP, an artificial bone analogs, in parallel. Both materials works well for this novel 3D cell culture model. Conclusion: This novel 3D culture model is a straight-forward and easy-to-observe model in order to understand the bone marrow microenvironment. It is also suitable to study the cell proliferation, differentiation, mobilization and homing, etc. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Mesenchymal stem cells (MSCs), like non-hematopoietic multipotent stem cells, are considered as the most promising stem cells for clinical use, such as treatment of aplastic anemia, graft-versus-host-disease after allogeneic hematopoietic stem cell (HSC) transplantation, as well as tissue engineering, etc. But the fate of MSC in vivo remains almost unknown, including its homing, proliferation and the interaction between MSC and surrounding cells. Target transplantation has been a dream of researchers for many years, especially for such important cells as MSC or HSC, and now it can be made possible by our self-established novel Magnetism-induced cell target transplantation (MagiC-TT). OBJECTIVE: To explore the distribution and survival of donor MSCs transplanted by Magnetism-induced cell target transplantation (MagiC-TT) in vivo, with dual fluorescent protein transgenic mice model. Methods and results: 1) Magnetized cells: the C57BL/6 RFP-MSCs were bought from Cyagen Biosciences Inc. (China) and were magnetized by self-made Au@Fe nano-particle, positive cells were sorted by MACS column. 2) Cell biology: Both magnetized and wild type (wt) cells were stained by Wright Giemsa and HE staining, as well as Prussian blue, there were no differences in cell morphology, while the particles of Au@Fe exist within or on the surface of magnetized cells. CCK8 method did not find any statistical significances in cell proliferation (P=0.802), cell cycle and cell viability. 3) In vitro study: In order to study the influence of magnetism to magnetized cells, cells' migration to magnetism and proliferation curve, transwell migration and matrigel migration assays were carried out. Within the magnetic field, magnetized cells can migrate through matrigel and transwell membrane much more efficiently, 174±22 vs. 2±1 per 200X microscopic vision (P Discussion: This study shows that magnetized MSCs have extra potential of moving under magnetism and comparable biology with wild-type cells. MagIC-TT can help MSCs' homing to bone marrow effectively, withdrawal of magnetic field permits MSCs to migrate into many tissues such as lungs, liver and spleen etc. Donor MSCs can survive for at least 3m in vivo. CONCLUSION: This dual transgenic mice model demonstrated that target cell transplantation can be achieved by MagIC-TT technology, and that it is useful in studying MSC and its mechanism in vivo. MagIC-TT also can be used in other cell therapy, by helping cells migrate to target tissues and organs in the future. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background Allogeneic hematopoietic stem cell transplantation is an effective method for treatment of hematological malignancies, while GVHD and graft rejection are main complications, which seriously affect patients' survival rates and quality of life. Aim Establishing allo-transplantation mice model with mRFP and GFP transgenic mice, to simulate clinical hematopoietic stem cell transplantation and explore the mechanism of stem cell homing and GVHD. Methods 1) Thirteen C57BL/6 GFP transgenic mice, used as recipients, were irradiated with 7 Gy. Each mouse was injected through caudal vein with 2*106 bone marrow cells isolated from FVB mRFP transgenic mice. 2) Symptoms like weight loss, depilation, diarrhea were observed as GVHD manifestation while survival rates were evaluated. Routine blood test and FACS were performed at different time points to confirm hematopoiesis reconstitution. 3) Mice were perfused with paraformaldehyde under anesthesia to fix the tissue, while pathological examination and real-time PCR were performed for studying donor and recipient cells interactions in different organs. 4) Semi-solid decalcification was used to treat the femora before observing under confocal microscope directly or after making frozen section, three-dimensional reconstruction were made to observe the cellular interaction, especially for cells within the bone marrow. Result 1) Depilation, wrinkled skin, hunchback and sharp decline of weight were observed in 8/13 mice. Routine blood test implicated hematopoietic reconstitution. FACS showed 86.1%±7.8% mRFP+ cells in peripheral blood of recipients. 2) mRFP+ cells were found distributing throughout the body's organs. mRFP+ Lymphocyte infiltration and inflammatory exudate were seen especially in the small intestine, lung, liver and skin (Fig.1). GFP+ cells were found surrounding mRFP+ cells in the bone marrow of the femora decalcified with semi-solid decalcification. Their interactions can be further observed clearly in bone marrow microenvironment in three-dimensional reconstruction by confocal microscope (Fig.2). Discussion Owing to RFP on donors' cells and GFP on recipients' cells, together with our novel protocol named semi-solid decalcification, we can visually observe the donor and recipient cells' location, ratio and cellular interaction, as well as morphological changes. Within various tissues especially for such tissues as bone marrow and lung, the details between cells can be studied lively by fluorescence microscope and confocal microscope. In recipient mice with GVHD, donor cells can be found in various target tissues such as intestine, lung, liver and skin. Gene marked cells with fluorescence protein can benefit morphological, immunological, cytogenetic and molecular studies in recipients after HSCT. Conclusion The allo-transplantation model with mRFP and GFP transgenic mice is powerful in study of Stem Cell Homing and Donor-Recipient Cellular Interaction. The cellular interaction can be easily observed by three-dimensional reconstruction after semi-solid decalcification, especially for bone marrow and lung. Disclosures: No relevant conflicts of interest to declare.

Abstract 4732 Background: Bone defection repairing is a good model to study the hematopoiesis and its environment, during which there are reformations of niches and marrow cavity, different cells' migrations and evolutions, etc. Tissue-engineering-bone (TEB) is a kind of scaffold that degrades gradually during bone reformation. In order to observe different cells growing within the rigid, opaque, friable and porous TEB, we established a novel technique named semi-solid decalcification (SSD). Aim: With the TEB repairing model, we hope to address 3 questions: Firstly, could a functional bone with marrow cavity be reformed with the TEB? Secondly, what are the contributions of cells within and outside the TEB? Thirdly, how do the hematopoietic cells cross-talking with the microenvironment during bone reformation? Method: 1. GFP+ mesenchymal cells were isolated from GFP transgenic FVB mice, expanded/purified with a murine MSC kit (cat no. MUCMX-90011, Saiye Company, China). 2. TEB b-TCP scaffold (3mm × 5mm, with microtubule pleural furrow, Bio-lu Company, France) was preloaded with 6th passage of eGFP mesenchymal cells (TEB-MC-GFP) 2d before implantation. 3. Five mRFP transgenic FVB mice were made segmental femur resects (5mm, half of femur length) by microsurgery, then 2 with internal fixation only as control, 3 were implanted with TEB-MC-GFP and internal fixation ( Fig 1 a, 1 b). 4. Bone formation throughout the defects was assessed radio graphically 4, 8 and 12w after the implantation. 5. Total body paraformaldehyde perfusion fixation was performed 12w after, and then samples of different organs were collected for frozen sections except the femur, while the femur underwent SSD before observation under confocal microscope and further frozen section. 6. Samples from different parts of the femurs were taken under invert fluorescence microscope after SSD, for detecting GFP and cytokines gene expression by RQ-PCR, ABL as control gene. Results: 1. X-ray shows the internal fixation is reliable and 5mm bone defects could not self-cure without TEB. 2. Completed cortical bone has covered the TEB repairing femur defects 12w after operation ( Fig 1 c). 3. Observing the frozen section under confocal microscope after SSD shows that: (a) eGFP+ mesenchymal cells grow adherently inside the pores of the TEB before implantation ( Fig 2 a, 2 b). (b) New cartilage and bone tissues had been formed, many eGFP+ and mRFP+ cells growing together in the pores of relic scaffold, new-forming collagen with red fluorescence inserting into the pores of TEB. To our surprising, eGFP+ cells are the majority component of marrow cavity. (c) Near rebuilt medullary canal, small “cabins” composed by eGFP+ and/or mRFP+ cells can been clearly observed, synapses and hairs of cells can be distinguished ( Fig 3 ). (d) Middle or small blood vessels around and within the TEB were constructed by mRFP+ and/or eGFP+ cells. eGFP+ cells could be found not only in muscles nearby, but also in murine kidney, lung and gut, et al. 4. The eGFP/ABL ratio of RQ-PCR in the middle of TEB > in the connection of TEB with normal bone > muscles nearby, which are 0.436±0.036, 0.140±0.038 and 0.098±0.024, respectively(P=0.000), in accordance with the expression of VEGF/ABL (P=0.003). Conclusion: This eGFP-to-mRFP transgenic mice femur repairing model was proved, at least in principle, to be efficient to mimic the reforming of bone marrow and stem cell niche. With the help of SSD, we can easily study the relationship and cross-talking of cell-cell and cell-matrix in a 3-D pattern, which is of the most important to understand the mechanism of hematopoiesis microenvironment. However, further researches are carrying on, to exclude the possible mixture of eGFP+ hematopoietic cells into mesenchymal cells before TEB-MC-GFP implantation and to better identify the subtypes of existing cells within the TEB. PS: Thanks to the support of NSFC (No.30901367, 31070866). Download : Download high-res image (119KB) Download : Download full-size image Download : Download high-res image (53KB) Download : Download full-size image Download : Download high-res image (135KB) Download : Download full-size image Disclosures: No relevant conflicts of interest to declare.

Abstract 2625 Background: It remains a huge challenge to observe fluorescent protein (GFP, RFP, etc.) gene-marked cells in bone, since bone is compact, poor lucency and porous, with different tissues such as vessels, nerve, cells, matrix and blood interlacing inside. However, it is very important to study the location, growth, migration and interaction of different stem cells and their offspring in bone. Aim: To better study different fluorescent protein gene-marked stem cells and microenvironment in bone, we establish a novel semi-solid decalcification (SSD) and research system. Methods: 1)Transplantation: Male BABL/C-GFP(H-2d) transgenic mice as donors and female FVB-RFP(H-2q) transgenic mice as recipients. Each RFP recipient mice were injected i.v. 5×10e6 allogeneic GFP bone marrow cells after 8 Gy TBI (n=10). Routinely, mice survival, weight, hemogram, GVHD manifestation were observed, with the fluorescence cells in peripheral blood and organs being traced. 2)Sections preparation: Total body perfusion fixation was performed with paraformaldehyde 21d after transplantation, and then different samples were collected for pathological examinations. The femurs were made frozen sections after semi-solid decalcification (SSD) system, while GMA plastic-embedding sections without decalcification, paraffin sections after EDTA decalcification, frozen sections after EDTA decalcification were also prepared as controls. Sections were observed by confocal microscopy. 3)Other researches: After SSD, observation and three-dimensional reconstruction were done by confocal microscopy; target tissue and cells were picked up for real-time quantified PCR for fluorescent protein genes and cell proliferation cytokines. Results: 1)Recipients RFP mice gained WBC recovery on (18.0±1.2)d, 90.0%±2.3% peripheral cells were GFP+ (n=10), 6 of 10 developed GVHD within 3m. 2)During SSD, hard component of the bone disappeared slowly, replaced gradually by semi-solid substance. SSD is even workable when the bone's diameter is large than 10cm. Frozen sections after SSD clearly showed unchanged position, form, and fluorescence of the GFP and RFP cells with repeatable hematoxylin and eosin(HE)and Wright-giemsa (RG) staining and immunohistochemical staining, fluorescence chromosomal in situ hybridization (FISH) after fluorescence observation and information from different tests of the same section can also be synthesized by computer. However, GMA cold embedding section could keep the cells where they are while losing the fluorescence, further more, embedding section only works well when the bone tissue is small (diameter Conclusion: The SSD system shows great potency for the research of stem cells in vivo in bone while maintaining the morphological characteristics and structures between different cells without losing fluorescence signals. Another fantastic advantage is that a large number of techniques can be combined to our system to help us understand the homing, growth, proliferation, differentiation, migration and interaction of different target stem cells and their offspring. Disclosures: No relevant conflicts of interest to declare.