MARCO+ Macrophage Dynamics in Regenerating Liver after 70% Liver Resection in Mice

Background: Macrophages play a key role in liver regeneration. The fates of resident macrophages after 70% resection are poorly investigated. In this work, using the MARCO macrophage marker (abbreviated from macrophage receptor with collagenous structure), we studied the dynamics of mouse liver resident macrophages after 70% resection. Methods: In BALB/c male mice, a model of liver regeneration after 70% resection was reproduced. The dynamics of markers CD68, TIM4, and MARCO were studied immunohistochemically and by using a Western blot. Results: The number of MARCO- and CD68-positive macrophages in the regenerating liver increased 1 day and 3 days after resection, respectively. At the same time, the content of the MARCO protein increased in the sorted macrophages of the regenerating liver on the third day. Conclusions: The data indicate that the number of MARCO-positive macrophages in the regenerating liver increases due to the activation of MARCO synthesis in the liver macrophages. The increased expression of MARCO by macrophages can be regarded as a sign of their activation. In the present study, stimulation with LPS led to an increase in the expression of the Marco gene in both Kupffer cells and macrophages of bone marrow origin.

Multipotent Stromal Cell Extracellular Vesicle Distribution in Distant Organs after Introduction into a Bone Tissue Defect of a Limb

When administered intravenously, extracellular vesicles derived from multipotent stromal cells (MSC EVs) immediately pass through the lungs along with the blood and regularly spread to all organs. When administered intraperitoneally, they are absorbed either into the blood or into the lymph and are quickly disseminated throughout the body. The possibility of generalized spread of MSC EVs to distant organs in case of local intratissular administration remains unexplored. However, it is impossible to exclude MSC EV influence on tissues distant from the injection site due to the active or passive migration of these injected nanoparticles through the vessels. The research is based on findings obtained when studying the samples of lungs, heart, spleen, and liver of outbred rabbits of both sexes weighing 3–4 kg at various times after the injection of EVs derived from MSCs of bone marrow origin and labeled by PKH26 into an artificially created defect of the proximal condyle of the tibia. MSC EVs were isolated by serial ultracentrifugation and characterized by transmission electron microscopy and flow cytometry. After the introduction of MSC EVs into the damaged proximal condyle of the tibia of rabbits, these MSC EVs can be found most frequently in the lungs, myocardium, liver, and spleen. MSC EVs enter all of these organs with the blood flow. The lungs contained the maximum number of labeled MSC EVs; moreover, they were often associated with detritus and were located in the lumen of the alveoli. In the capillary network of various organs except the myocardium, MSC EVs are adsorbed by paravasal phagocytes; in some cases, specifically labeled small dust-like objects can be detected throughout the entire experiment—up to ten days of observation. Therefore, we can conclude that the entire body, including distant organs, is effected both by antigenic detritus, which appeared in the bloodstream after extensive surgery, and MSC EVs introduced from the outside.

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

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.

Bone marrow-derived neural crest precursors improve nerve defect repair partially through secreted trophic factors

Background Emerging evidence suggests that neural crest-derived cells (NCCs) present important functions in peripheral nerve regeneration to correct the insufficiency of autogenous Schwann cells. Postmigratory NCCs have been successfully isolated from adult rat bone marrow in our previous work. In this study, we aim to provide neural crest-derived Schwann cell precursors (SCPs) for repair of nerve defects in adult rats, and partially reveal the mechanisms involved in neuroregeneration of cell therapy. Methods A clonal cell line of neural crest precursors of rat bone marrow origin (rBM-NCPs) with SCP identity was expanded in adherent monolayer culture to ensure the stable cell viability of NCPs and potentiate the repair of nerve defects after rBM-NCPs implantation based on tissue engineering nerve grafts (TENG). Here the behavioral, morphological, and electrophysiological detection was performed to evaluate the therapy efficacy. We further investigated the treatment with NCP-conditioned medium (NCP-CM) to sensory neurons after exposure to oxygen-glucose-deprivation (OGD) and partially compared the expression of trophic factor genes in rBM-NCPs with that in mesenchymal stem cells of bone marrow origin (rBM-MSCs). Results It was showed that the constructed TENG with rBM-NCPs loaded into silk fibroin fiber scaffolds/chitosan conduits repaired 10-mm long sciatic nerve defects more efficiently than conduits alone. The axonal regrowth, remyelination promoted the reinnervation of the denervated hind limb muscle and skin and thereby alleviated muscle atrophy and facilitated the rehabilitation of motor and sensory function. Moreover, it was demonstrated that treatment with NCP-CM could restore the cultured primary sensory neurons after OGD through trophic factors including epidermal growth factor (EGF), platelet-derived growth factor alpha (PDGFα), ciliary neurotrophic factor (CNTF), and vascular endothelial growth factor alpha (VEGFα). Conclusions In summary, our findings indicated that monolayer-cultured rBM-NCPs cell-based therapy might effectively repair peripheral nerve defects partially through secreted trophic factors, which represented the secretome of rBM-NCPs differing from that of rBM-MSCs.

Implantation of the collagen membrane with adsorbed multipotent stem cells in experiment

Objective. To follow-up the processes, taking place with autologous multipotent mesenchymal stem cells (ММSC) of the bone marrow origin after their implantation into the organism on surface of the collagen material in experiment. Маterials and methods. The changes in subcutaneous adipose cellular tissue in rats after implantation of the collagen membrane with passively adsorbed on the MMSC on the surface with transfected gene GFP and additionally stained cellular membranes with Vybrant® CM-Dil were studied up, using the light microscopy methods and a luminescence. Results. While application of a luminescence in a one week after implantation of the collagen material with the MMSC adsorbed in a neighborhood with the implant there were revealed typical very small vessels owing a one-layer membrane, which have fluoresced intensely while a rodamine filter was installed. On the second week on periphery of the collagen material and in neighborhood to the implant on background of a rodamine filter application only small inclusions inside macrophages were shining with a red color. Subsequently step by step the quantity of such shining objects have reduced down to solitary ones, presenting very rarely, as well as their fluorescence intensity. Conclusion. The main cause for the tissue vascularization enhancement around the implanted collagen with the MMSC adsorbed on the surface is their differentiation into cellular elements of vascular membranes. Starting from the second week all the MMSC undergo destruction and are phagocytized by macrophages, also in this term the structures from these MMSC disappear as a result of their differentiation, including the vessels.

DECREASE IN ACTIVITY OF THE INFLAMMATORY PROCESS AFTER IMPLANTATION OF SILICONE WITH ADSORBED MULTIPOTENT STROMAL CELLS

Background. The wide spread of cellular technologies will sooner or later lead inevitably to the introduction of MMSC or their exosomes to patients who have implanted artificial materials in their organism. The engraftment of foreign bodies is accompanied first by acute and then chronic inflammation, very often acquiring a granulomatous character. A decrease in the activity of the inflammatory response can theoretically improve the results of implantation.Objective. To study the results of the influence of autologous MMSC of bone marrow origin (AMMSCBMO) adsorbed on silicone, on the inflammatory process that accompanies the implantation of this polymer in theexperiment. Design and methods. In different times, the condition of tissues around the implanted silicone with adsorbed AMMSCBMO was studied by method of light microscopy.Results. After the implantation of silicone with adsorbed AMMSCBMO, as compared with the results of the introduction of this material without use of cellular technologies, a smaller volume of loose fibrous connective tissue is formed around the encapsulated polymer. In this connective tissue after AMMSCBMO use the absolute number of all cells is lower in 2 weeks, and the percentage and numerical density of neutrophils — within 1–2 weeks. On the contrary, the relative number of erythrocytes within 1–2 weeks and macrophages at 1–3 weeks under the conditions of AMMSCBMO use were greater.Conclusion. A smaller volume of loose fibrous connective tissue and a decrease in the severity of cellular infiltration, in particular, the number of neutrophils, around the implanted silicone with AMMSCBMO indicates a decrease in the activity of the inflammatory reaction as a result of the use of cellular technologies, more rapid cleansing of postoperative wound from debris, the success of reparative processes and the creation of conditions for the integration of a foreign bodies into the organism.

Label-free density-based detection of adipocytes of bone marrow origin using magnetic levitation

The first report on application of magnetic levitation technology for detection of adipogenic cells based on single cell density measurement.

Label-Free Density-Based Detection of Adipocytes of Bone Marrow Origin Using Magnetic Levitation

ABSTRACTAdipocyte hypertrophy and hyperplasia are important parameters in describing abnormalities in adipogenesis that are concomitant to diseases such as obesity, diabetes, anorexia nervosa and osteoporosis. Therefore, technical developments in detection of adipocytes become an important driving factor in adipogenesis research. Current techniques such as optical microscopy and flow cytometry are available in detection and examination of adipocytes, driving cell- and molecular-based research of adipogenesis. Even though microscopy techniques are common and straightforward, they are restricted for manipulation and separation of the cells. Flow cytometry is an alternative, but mature adipocytes are fragile and cannot withstand the flow process. Other separation methods usually require labeling of the cells or usage of microfluidic platforms that utilize fluids with different densities. Magnetic levitation is a novel label-free technology with the principle of movement of cells towards the lower magnetic field in a paramagnetic medium depending on their individual densities. In this study, we used magnetic levitation device for density-based single cell detection of differentiated adipogenic cells in heterogeneous populations. Results showed that magnetic levitation platform was sensitive to changes in lipid content of mesenchymal stem cells committed to adipogenesis and it could be successfully used to detect adipogenic differentiation of cells.