Post transplant administration of AMD3100 mobilizes recipient residual HSCs, increasing marrow niche availability.

<p>Lethally irradiated C57BL/6 CD45.2 Thy1.2 mice (10.5 Gy) were injected via tail vein with sorted KTLS hematopoietic stem cells (2500 cells/mouse) obtained from C57BL/Ka Thy1.1 donor mice. At 48 hours after transplantation, recipient mice were injected subq with a single dose of AMD3100 at 5 mg/kg or PBS control. Twenty-four hours after this injection, the mice were sacrificed, and the 2 femurs and the spleen were harvested. Recipient-origin stem cells (CD45.2⁺ KLS cells) and donor derived stem cells (CD45.1⁺ KLS cells) in the spleen and in the marrow were determined. Marrow CFUs-GM and Bursting forming units of erythrocytes (BFUs-E) were measured using Methocult® GF 3434 (1×10⁵ cells/dish, triplicate dishes per individual mouse). Percent donor stem cell homing efficiency to the spleen was calculated by dividing the absolute number of CD45.1⁺ KTLS stem cells in the recipient's spleen by the number of injected KTLS stem cells. The graphs are representative of 2 separate sets of experiments with 5–10 mice in each group of each experiment. A: total number of recipient CD45.2⁺ KLS stem cells in 2 femurs. B: marrow CFU-GM and BFU-E assay (numbers of donor CFU-GM and BFU-E/3×10⁵ marrow cells). C: total number of recipient stem cells in the spleen. D: % donor stem cell homing efficiency in the spleen. E: Measurement of hematopoietic stem/progenitor cell mobilization after prolonged AMD3100 administration. Transplanted mice that were given AMD3100 at 5 mg/kg body weight or PBS subcutaneously every other day beginning at day +2 for 60 days and naïve normal control mice were injected with AMD3100 subcutaneously at 5 mg/kg body weight or PBS. Blood samples were collected immediately prior to the injection and 4 hours after injection, and colony-forming units and white blood cell counts determined. Colony-forming units were represented as colony numbers/30 µl blood (n = 5 in each group. Open bar: before AMD3100 injection. Filled bar: 4 hours after AMD3100 injection). *: p<0.05. NS: not statistically significant.</p

Post transplant administration of AMD3100 improves animal survival in a murine transplantation model.

<p>KTLS cells from C57BL/Ka CD45.1 Thy1.1 mice were injected via tail vein into lethally irradiated C57BL/6 CD45.2 Thy1.2 mice (250 KTLS cells per mouse). Beginning 2 days after transplant (day +2), the mice were injected subcutaneously with PBS control or AMD3100 at 5 mg/kg body weight every Monday, Wednesday, and Friday until day +56. Animal survival was monitored daily. In experiment #1 and #2, the mice received 10.5 Gy total body irradiation using a Cesium irradiator. In experiment #3, the mice received 9.5 Gy total body irradiation. (n = 10 mice in each group in each experiment).</p

Schematic diagram of AMD3100's mechanisms of action.

<p>Post transplant administration of AMD3100 likely has 3 effects. <u>1</u>. AMD3100 mobilizes residual recipient's radioresistant HSCs (indicated as red circle), thus freeing up more niches for healthy donor HSCs (indicated as purple circles) to engraft. This is supported by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011316#pone-0011316-g005" target="_blank"><b>Fig. 5</b></a>. <u>2</u>. AMD3100 relieves the inhibitory effect of SDF-1 on primitive HSCs, allowing for engrafted HSCs to proliferate. <u>3</u>. AMD3100 mobilizes engrafted donor HSCs and mobilized HSCs are more active and more proliferative. Effects <u>2</u> and <u>3</u> are supported by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011316#pone-0011316-g006" target="_blank"><b>Fig. 6</b></a>. Recipient HSCs after irradiation likely have survival disadvantage compared to donor HSCs. The end result is selective engraftment and expansion of donor hematopoietic cells.</p

Post transplant administration of AMD3100 selectively enhances reconstitution of all donor-derived hematological lineages of cells.

<p>KTLS cells from C57BL/Ka CD45.1 Thy1.1 mice were injected via tail vein into lethally irradiated C57BL/6 CD45.2 Thy1.2 mice (250 KTLS cells per mouse). Beginning 2 days after transplant (day +2), the mice were injected subcutaneously with PBS control or AMD3100 at 5 mg/kg body weight every Monday, Wednesday, and Friday until day +56. Hematological recovery and cell subset analyses were determined at the time points indicated, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011316#s4" target="_blank">Methods</a>. The graphs are representative of 3 separate sets of experiments with 10 mice in each group in each experiment. A: Blood nucleated cell counts (/µl) (normal range of mouse white cell counts: 1.8–10.7×10³/µl). B: Donor-derived CD45.1⁺ cell counts (/µl). C: Blood recipient origin CD45.2⁺ cell counts (/µl). D: Blood CD3⁺ T cell counts (/µl). E: Blood B220⁺ B cell counts (/µl). F: Hemoglobin concentration (g/dl) (normal range of mouse hemoglobin level: 11–15.1 g/dl). G: Blood platelet counts (×10⁶/ml) (normal range of mouse platelet count: 592–2972×10⁶/ml). *: p<0.05</p

Post transplant administration of AMD3100 increases CFUs-spleen and reduces cytokine, chemokine, and growth factor levels.

<p>A: CFUs-spleen assay. Lethally irradiated CD45.2 mice (10.5 Gy) were injected with whole marrow cells from CD45.1 mice (1×10⁵ marrow cells per mouse). The recipient mice were injected subcutaneously with PBS buffer or AMD3100 at 5 mg/kg every other day beginning at day +2. At day +9, the mice were sacrificed and CFUs-spleen measured (n = 5–10 mice per group). B: Plasma cytokine/chemokine measurement. Lethally irradiated CD45.2 mice (10.5 Gy) were injected with sorted KTLS stem cell from CD45.1 mice (7000 KTLS cells per mouse). The recipient mice were injected subcutaneously with PBS buffer or AMD3100 at 5 mg/kg every other day beginning at day +2. The mice were bled at Day +7 and plasma prepared. Plasma cytokines and chemokines (pg/ml) were measured as per manufacturer's instruction (Bioplex, Bio-Rad Laboratories) (n = 5 mice per group). G-CSF level was shown in a separate panel. C: 9 cytokines/chemokines that were significantly reduced in AMD3100-treated mice. D: Survival rate in lethally irradiated mice without HCT. CD45.2 mice were lethally irradiated (10.5 Gy) but not transplanted with HSCs. The mice were then administered subcutaneously with PBS or AMD3100 at 5 mg/kg every other day beginning at day +2 post irradiation until the end of experiments. Animal survival was monitored daily (data representative of 3 separate sets of experiments with 10 mice in each group of each experiment).</p

Post transplant administration of AMD3100 enhances donor cell marrow engraftment.

<p>The mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011316#pone-0011316-g001" target="_blank"><b>Fig. 1</b></a> were sacrificed at Day +65 post transplantation, and 2 femurs and 2 tibias were harvested and processed and analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011316#s4" target="_blank">Methods</a>. A: CFU-GM assay. CFU-GM colonies were measured in individual mice using MethoCult® GF 3434 mouse colony-forming cell assay. B: Histology. Bone marrow from individual mouse was examined by microscopy (4X: top panel and 100X: bottom panel) after decalcification and H/E staining. Trabecular bony structure, osteoblasts, hematopoietic cells, sinusoid vessels and acellular areas filled with lipid droplets were indicated. Normal mice did not receive transplant or AMD3100 injection. C: Total nucleated cell number from 1 femur and 1 tibia. D: Donor-derived CD45.1⁺ KLS stem cell number from 1 femur and 1 tibia of individual mouse. E: Recipient-origin CD45.2⁺ KLS stem cell number from 1 femur and 1 tibia of individual mouse. *: p<0.05.</p

Post transplant administration of AMD3100 increase donor cell division.

<p>A: In vivo cell division measurement. CD45.1⁺ KTLS cells were labeled with CFSE and injected into lethally irradiated C57BL/6 CD45.2 Thy1.2 mice (7×10³ KTLS cells/mouse). The 2 panels on the left showed the sorted CFSE-labeled KTLS cells prior to injection to recipient mice. The recipient mice were treated with AMD3100 at 5 mg/kg or PBS every other day beginning at day +2. The mice were sacrificed at day +7 and bone marrow cells measured for CFSE intensity (The 4 panels on the right. Top 2 panels: recipient mice treated with PBS control; Lower 2 panels: recipient mice treated with AMD3100). The CFSE intensity was gated on CD45.1⁺ donor derived cell population (n = 5 mice in each group). <b>B: In vitro marrow cell proliferation responses to AMD3100 or G-CSF.</b> Marrow cells (5×10⁵/well) were cultured with various concentrations of AMD3100 or recombinant G-CSF, and [³H] thymidine incorporation was measured (three separate sets of experiments with triplicates in each experiment).</p

Long-Term In Vivo Imaging of Multiple Organs at the Single Cell Level

<div><p>Two-photon microscopy has enabled the study of individual cell behavior in live animals. Many organs and tissues cannot be studied, especially longitudinally, because they are located too deep, behind bony structures or too close to the lung and heart. Here we report a novel mouse model that allows long-term single cell imaging of many organs. A wide variety of live tissues were successfully engrafted in the pinna of the mouse ear. Many of these engrafted tissues maintained the normal tissue histology. Using the heart and thymus as models, we further demonstrated that the engrafted tissues functioned as would be expected. Combining two-photon microscopy with fluorescent tracers, we successfully visualized the engrafted tissues at the single cell level in live mice over several months. Four dimensional (three-dimensional (3D) plus time) information of individual cells was obtained from this imaging. This model makes long-term high resolution 4D imaging of multiple organs possible.</p> </div

Ear-tissue can be visualized at the cellular level in living animal.

<p>(a) This picture showing the location of an engrafted heart graft in the ear pinna. (b) Skeletal muscle and small intestine tissues from EGFP mice were subcutaneously transplanted into the ear pinnae of BALB/c nude mice. The images were obtained 9 (muscle) and 22 (intestine) weeks after transplantation. The images were taken 107 µm (muscle) deep from surface. The depth for intestine could not be determined. (c) Heart and kidney tissues from EGFP mice were subcutaneously transplanted into the ear pinnae of BALB/c nude mice. Rhodomin B conjugated dextran was injected i.v. to visualize the blood vessels. The images were obtained 9 (heart) and 2 (kidney) weeks after transplantation. Green = EGFP; red = dextran. (d) EGFP⁺ neonatal thymic tissue was subcutaneously transplanted into the ear pinnae of BALB/c nude mice. Eight weeks later, the mice were irradiated and transplanted with DsRed⁺ T cell depleted bone marrow cells. The image was taken 4 weeks after bone marrow transplantation at the depth of 103 µm. Green = EGFP; red = DsRed. Scale bar = 100 µm.</p

Visualizing radiation-induced thymocyte apoptosis in ear-thymus graft.

<p>A nude BALB/c chimera containing DsRed⁺ hematopoietic cells were transplanted with a thymus from a B6 CD45.1 neonatal mouse (<48 hours old) into the ear pinna. Five weeks later, the ear pinna containing thymus graft was irradiated with 8.5 Gy. Cell apoptosis was then followed over time by in vivo two-photon imaging after injection of FAM-FLIVO. The images were taken 113 to 137 µm deep from surface. Representative pictures from each group are shown. The percentages of apoptotic cells are shown. Similar experiments have been repeated for three times. Green = EGFP; red = apoptotic cells. Scale bar = 50 µm.</p