Possible Kidney Complications after Application of Cell Technologies for the Repair of the Resected Liver.

The state of rat kidneys after injection of bone marrow multipotent stromal cells (MSC) labeled with Vybrant CM-Dil into intact or resected liver was studied by fluorescence microscopy. The main structural changes in the kidneys after MSC injection into intact and partially resected liver manifested as granular dystrophy and necrobiotic/necrotic changes in single epithelial cells of the distal tubules and collecting ducts, thrombosis of some vessels, progression of an ascending urinary tract infection (detection of dust-like fluorescent objects), which can be due to the immunomodulating or even immunosuppressive influence of MSC and their detritus. MSC injected into the intact or resected liver, as well as the products of their degradation were not detected in the kidneys at all terms of observation. After injection of MSC into partially resected liver, manifestations of bacterial contamination of the renal medulla appeared later. The injection of MSC into the liver can be complicated by thrombosis of the renal vessels, which should be taken into account when using this administration route in the cell therapy.

Dissemination of Multipotent Stromal Cells in the Organism after Their Injection into Intact and Resected Liver in the Experiment.

The possibility of dissemination of bone marrow multipotent stromal cells stained with Vybrant CM-Dil after injection into an intact and resected liver was studied using luminescence microscopy. Labeled cells were found in the kidneys, spleen, lungs, axillary, mesenteric, and inguinal lymph nodes. We observed dissemination of multipotent stromal cells and their detritus throughout the body that occurred only after filtration in the lungs, where most cells underwent destruction. Perivascularly located macrophages in various organs can phagocytize multipotent stromal cells and their detritus from blood vessels. The content of objects labeled with Vybrant CM-Dil in distant organs was significantly lower after multipotent stromal cell injection into the resected liver, which was associated with the deposition of cells in the damaged area of the organ and their partial entry into the abdominal cavity.

Injection of Multipotent Mesenchymal Stromal Cells as a Cause of Hemorrhages in the Regional Lymph Nodes: Experimental Study.

Hemorrhagic changes after subcutaneous injection of autologous bone marrow multipotent mesenchymal cells with transfected GFP gene and additionally stained cell membranes to WAG rats in the projection of ligated femoral vein were studied by fluorescent microscopy. Hemorrhages in tissues with experimental acute local venous occlusion were caused by a combination of venous hypertension with inflammation around the foreign body - the ligature used for ligation of the vein. Fibrin found in tissues together with erythrocytes in the hemorrhages could stimulate the formation of granulations and new vessels instead of damaged or thrombosed ones. Multipotent mesenchymal stromal cells and their detritus getting into the regional lymph nodes initiated immune reactions morphologically confirmed by stubborn hypertrophy and hyperplasia of the lymphoid nodules, hemorrhages, and manifest diapedesis of erythrocytes to the organ parenchyma and sinus system.

Some Peculiarities of Local Distribution of Multipotent Mesenchymal Stromal Cells after Their Injection into Intact Muscle Tissue in Experiment.

Changes in the muscular tissue after subcutaneous injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and additionally stained with cell membrane dye Vybrant CM-Dil in the projection of ligated femoral vein were studied by light microscopy with luminescence. Stromal cells injected through the skin can appear not only in the damaged tissue where acceleration of regeneration processes is required, but also in intact structures located in superficial or deeper layers. In intact muscular tissue, stromal cells spreading in the perivascular tissue initiate inflammation and migration of macrophages, activate and even trigger sclerotic processes due to differentiation into connective tissue cells (fibroblasts) and stimulation of proliferation and collagen synthesis by host fibroblasts. Injected multipotent mesenchymal stromal cells are gradually phagocytized by macrophages.

Results of Experimental Ligation of the Main Vein with the Use of Cell Technologies.

Autologous multipotent mesenchymal stromal cells (MMSC) of bone marrow origin with transfected GFP gene and additionally stained cell membranes were injected to rats through the skin in the projection of ligated femoral vein. The results were evaluated by fluorescent microscopy. No signs of MMSC incorporation into the wall of ligated vessel or reorganized collaterals were detected. Angiogenesis processes involving MMSC were detected in experimental rats within just 4 days and progressed until week 2 postinjection, mainly in granulations at the site of surgical intervention and the cicatrix forming there. Injected MMSC completely formed all tunics of the new vessels and incorporated in the vessels forming from the recipient cells. MMSC and the objects created from them were gradually eliminated with participation of macrophages and replaced by structures formed from the recipient cells.

Correction to: Possibility of Aggravation of Tissue Sclerosis after Injection of Multipotent Mesenchymal Stromal Cells Near the Forming Cicatrix in the Experiment.

The author name G. A. Chastikina should read G. A. Chastikin.

Possibility of Aggravation of Tissue Sclerosis after Injection of Multipotent Mesenchymal Stromal Cells Near the Forming Cicatrix in the Experiment.

The peculiarities of tissue sclerosis after injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and stained with Vybrant CM-Dil cell membrane dye were studied by light microscopy with luminescence. The surgical intervention consisting in ligation of the great vein was followed by tissue sclerotic transformation caused by direct damage and chronic inflammation caused by the presence of slowly resorbed ligature. Injection of stromal cells after this intervention led to formation of more extensive scar. This can attest to the possibility of stromal cells differentiation into connective tissue cells, fibroblasts, and stimulation of proliferation and collagen synthesis by host fibroblasts. A decrease in the volume of dense fibrous connective tissue due to scar reorganization at latter terms cannot not excluded.

Initial Stages of Angiogenesis after Acute Experimental Local Venous Outflow Disturbances and Application of Cell Technologies.

The initial stages of angiogenesis in rats after transcutaneous injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and stained cell membranes in the projection of ligated femoral vein were studied by fluorescent light and confocal laser microscopy. Large clusters of brightly fluorescing elongated fibroblast-like cells were seen in the paravasal tissue and in the postoperative scar and signs of angiogenesis were noted as soon as in 4 days. The injected cells not only formed new vessels, but also integrated into vessels formed by host cells. Some injected cells were phagocytizied by macrophages and the latter started to fluoresce due to the presence of the membrane dye. These macrophages within the specified period appeared in the regional inguinal lymph nodes where they formed clusters in the lymphoid parenchyma of the cortical substance.

[SOME REACTIONS OF THE REGIONAL LYMPH NODES OF RATS AFTER IMPLANTATION OF MULTIPOTENT STROMAL CELLS ADSORBED ON POLYHYDROXYALKANOATE INTO A BONE TISSUE DEFECT].

The reactions of the regional lymph nodes, caused by implantation of the autologous multipotent stromal cells of bone marrow origin (AMSCBMO) to accelerate the healing of mandibular bone defect were studied by fluorescent microscopy in inbred male Wag rats aged 6 months (n=62). After the introduction of polyhydroxyalkanoate transplant containing adsorbed AMSCBMO with a transfected Green Fluorescent Protein (GFP) gene into a damaged bone area, the lymphoid nodules in submandibular lymph nodes demonstrated the appearance of numerous large macrophages containing multiple oval fluorescent inclusions in the cytoplasm. The number of these macrophages increased within 2 weeks after surgery and then began to decline. Apparently, AMSCBMO introduced in this way, were partially absorbed by macrophages. After destruction of the structures formed from AMSCBMO, the debris was also phagocytized by macrophages. In either case, these macrophages appeared in the germinal centers of lymphoid nodules in lymph nodes, where the induction of immune responses against DNA and GFP protein was probable.

[ANGIOGENESIS IN THE TISSUES AFTER THE INJECTION OF STROMAL STEM CELLS OF BONE MARROW ORIGIN CLOSE TO THROMBOSED VEIN IN AN EXPERIMENT].

The effects of the injection of autologous multipotent stromal stem cells of bone marrow origin (MSSCBM) (mesenchymal stem cells) with green fluorescent protein gene, additionally marked with DAPI nuclear stain, close to a thrombosed hindlimb vein, were studied by fluorescent microscopy in adult male Wag rats (n = 214). The control groups consisted of intact rats (n = 12), animals with venous thrombosis without the injection of MSSCBM (n = 71) and rats that received paravasal injection of MSSCBM, but without preliminary modeling of venous thrombosis (n = 72). It was found that MSSCBM participated in the development of granulation tissue at the site of surgical intervention performed during the modeling of thrombosis. The rapid development of granulation tissue at the site of surgical trauma may contribute to faster wound clearance from detritus, nonviable tissue and antigenic substances, early onset of tissue repair processes and rapid healing. Restoration of blood flow in the tissue region of a thrombosed vein began later than after the intravenous injection of MSSCBM.

[The rat submandibular lymph node after introduction in mandibular bone defect ultipotent mesenchymal cells adsorbed on polyhydroxyalkanoate scaffold].

The reactions of rat regional lymph nodes, caused by implantation of the autologous multipotent mesenchymal stromal cells of a bone marrow origin (AMMSCBM) for acceleration of bone defect regeneration in bottom jaw experiment were studied by methods of fluorescent light microscopy. After introduction in an injury site of a bottom jaw bone of polyhydroxyalkanoate with adsorbed AMMSCBM with a transfected GFP gene the numerous large macrophages with a set of oval fluorescent inclusions in cytoplasm appear in lymph nodules of submandibular lymph nodes. The number of such macrophages increases within 2 weeks after operation, and further starts decreasing. Probably, entered via such way the AMMSCBM partially are phagocytized by macrophages. At destruction of the structures created from AMMSCBM, debris also are phagocytized by macrophages. In that and other case these macrophages appear in the germinative centers of lymph nodules in lymph nodes where initiation of immunity reactions against DNA and same GFP isn't excluded.