Some Special Aspects of Liver Repair after Resection and Administration of Multipotent Stromal Cells in Experiment

Abstract

Changes in rat liver after resection and injection of autologous multipotent mesenchymal stromal cells of bone marrow origin (MSCs) transfected with the GFP gene and cell membranes stained with red-fluorescent lipophilic membrane dye were studied by light microscopy. It was found that after the introduction of MSCs into the damaged liver, their differentiation into any cells was not found. However, under the conditions of MSCs use, the number of neutrophils in the parenchyma normalizes earlier, and necrosis and hemorrhages disappear more quickly. It was concluded that the use of MSCs at liver resection for the rapid restoration of an organ is inappropriate, since the injected cells in vivo do not differentiate either into hepatocytes, into epithelial cells of bile capillaries, into endotheliocytes and pericytes of the vascular membranes, into fibroblasts of the scar or other connective tissue structures, or into any other cells present in the liver.

Keywords: liver; liver resection; macrophages; multipotent mesenchymal stromal cells; neutrophils.

Figure 1

Liver of rats at various times after resection. Staining with hematoxylin and eosin: (a) After 1 week at the site of resection and injection of MMSCs, there is a wide forming scar extending deep into the parenchyma. The scar tissue contains many leukocytes, very large, more than 50 μm, cells with a foamy cytoplasm and structureless eosinophilic fragmented detritus; (b) a capsule of dense connective and fibrous tissue with a high content of macrophages was restored at the site of the operation with the subsequent introduction of MMSCs by 4th week. Thin layers of connective tissue containing groups of vessels and young bile capillaries depart from the scar on the surface into the parenchyma; (c) on the 2nd week after surgery, without MMSCs injection, some peripheral veins are thrombosed, there are many neutrophils in the thrombi; (d) after resection without the introduction of MMSCs, even after 5 weeks, there are many neutrophils in the parenchyma and vessels; (e) 4 weeks after damage and injection of MMSCs, areas of plasma impregnation with necrosis of hepatocytes remain in the parenchyma; (f) hemorrhage with plasma impregnation of the parenchyma and necrosis of adjacent cells on 4th week after resection and injection of MMSCs.

Figure 2

Results of a study using luminescence and immunohistochemistry of animal liver after resection and injection of MMSCs: (a) After 1 week in the developing scar on the surface of the organ, there are many objects with very bright fluorescence when using a rhodamine filter. In the layer of connective tissue that goes deep into the liver, there are no such objects. The result of computer alignment of images obtained using the Alexa 488 and rhodamine filters; (b) in the 1st week, the structures of the scar contain many cells of the macrophage series. There is a clear border between the macrophage scar and the preserved parenchyma. Reaction with antibodies against CD68 antigen, staining with diaminobenzidine and hematoxylin; (c) after 1 week, far from the operation site, near the peripheral vessels, there are groups of cells with very intense fluorescence when a rhodamine filter is installed. The result of computer alignment of images obtained using the Alexa 488 and rhodamine filters; (d) after 2 weeks, at a distance from the resection site, under the conditions of using a rhodamine filter, the cells located near a large vessel glow brightly. The result of computer alignment of images obtained using the Alexa 488 and rhodamine filters; (e) in the 3rd week, next to the bifurcated vessel, there is a large cell (about 20 microns) with a dark nucleus, some of its numerous cytoplasmic inclusions fluoresce brightly when using a rhodamine filter, while others do so when installing an Alexa 488 filter. The result of computer alignment of images obtained using Alexa 488 and rhodamine filters; (f) in the restored capsule on the surface of the organ, after 5 weeks, cells of various sizes and shapes intensively fluoresce in different colors. The result of computer alignment of images obtained using the Alexa 488 and rhodamine filters; (g) a thin layer of connective tissue at a distance from the operation site in the 5th week contains wide vessels and cells with brighter fluorescence against the background of the application of a rhodamine filter. The result of computer alignment of images obtained using the Alexa 488 and rhodamine filters; (h) after 5 weeks, a pronounced positive reaction to the histiocytic antigen was noted in the cells of a thin layer of connective tissue at a distance from the site of damage. Macrophages are located in one row and sometimes completely surround other cells that are not hepatocytes. Reaction with antibodies against CD68 antigen, staining with diaminobenzidine and hematoxylin.