Fig 2: Network analysis of liver transcripts post pig CSF1-Fc treatment. Expression data for mouse livers +/- CSF1-Fc (n = 3) was analyzed alongside that of BMDM expression data (+/- LPS). (a) A network graph of transcript-to-transcript Pearson correlation relationships was filtered to show relationships of r ≥ 0.96, resulting in a graph of 2,555 nodes (transcripts) connected by 265,108 edges (Pearson correlation relationships). The graph was then clustered using the MCL clustering algorithm into groups of co-expressed genes. Nodes with the same color belong to the same cluster of co-expressed genes and tend to be highly connected within the network. (b) Expression data for five clusters with each point on the graph representing an individual mouse.

Fig 3: Effect of pig CSF1-Fc on BM cells and bone. Mice were injected with PBS or 1 µg/g pig CSF1-Fc for four days prior to sacrifice on day 5. The right femur from each mouse was harvested and prepared for histological examination. All graphs show the mean ± SEM. Significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001 using a Mann–Whitney test. (a) TRAP IHC was performed. Black arrows represent OCL in epiphyseal plate. The number of positive cells in each section was counted. n = 4 mice/group. (b) The myeloid:erythroid ratio was determined and graphed. n = 4 mice/group. (c) BM cells were flushed from the femurs post-mortem and prepared for FACS as described in materials and methods. The percentage of F4/80 and Gr1 cell populations were determined by exclusion of dead cells using Sytox blue. n = 12 mice/group.

Fig 4: Pig CSF1-Fc produces viable CSF1-dependent proliferation in vitro and has extended plasma half-life in vivo. (a) CSF1-Fc molecule was produced by CSF1 joined to the hinge-CH3 region of pig IgG1a. (b) CSF1-dependent Ba/F3pCSF1R cells were cultured in rh-CSF1, harvested, washed twice in PBS, and plated for the optimized cell viability assay with either pig CSF1 or pig CSF1-Fc for 48 hours. Pig BM cells were flushed from an adult pig rib and placed in culture with either pig CSF1 or pig CSF1-Fc for 48 hours. Following addition of MTT solution and solubilization, optical density was read at 570nm using a plate reader. Results are the average of triplicate determinations ± SEM from three experiments. (c) Three weaner pigs were injected with either 0.5 mg/kg or 1.2 mg/kg pig CSF1 or Fc CSF1-Fc respectively and blood collected at time points above for CSF1 and Fc CSF1-Fc levels to be determined by ELISA. The mean ± SEM is graphed.

Fig 5: Effect of CSF1-Fc on body weight, organ weights, and white blood cell counts. Mice were injected with PBS or 1 µg/g pig CSF1-Fc for four days prior to sacrifice on day 5. Blood was collected into EDTA tubes post-mortem and complete blood count assessment performed. Graphs show the mean ± SEM. Significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001 using a Mann–Whitney test. n = 20 mice per group for weights and n = 12 mice per group for blood cell counts (a) Body weight was recorded before each injection. Total body weight change over the duration of the experiment was graphed (b) Spleen/body weight ratio (c) Liver/body weight ratio (d) Total WBC count (e) Monocyte number (f) Neutrophil number (g) Lymphocyte number. PBS, phosphate-buffered saline.