Experimental osteoporosis and its correction.

Osteoporosis was modeled in rats by chronic (6 months) treatment with omeprazole or serotonin, and bone tissue status was studied in experimental hepatic fibrosis and during serotonin treatment under conditions of hepatic fibrosis. Serum levels of calcium, phosphorus, alkaline phosphatase, albumins, and creatinine and bone tissue levels of calcium, phosphorus, magnesium, and iron were measured. Treatment with mesenchymal stem cells over 6 months reduced the severity of osteoporosis.

Phenotypic differences between mdx black mice and mdx albino mice. Comparison of cytokine levels in the blood.

In mdx mice, mutation in the muscle protein dystrophin gene results in the development of chronic degeneration of the muscle tissue. We performed a comparative analysis of blood cytokine levels in mdx mice, classical black mice and mice with additional genetic defect responsible for the manifestations of oculocutaneous albinism. In mdx albino mice, the total pool of cytokines (IL-10, IL-6, IL-5, IL-2, IL-1α, IL-4, IL-17, granulocyte-macrophage growth factor, TNF-α, and IFN-γ) was increased. This increase was not associated with selective release of one of the above cytokines into the blood. The fraction of pro-inflammatory cytokines (IL-6, IL-1α, TNF-α) was increased in the total pool and the percentage of antiinflammatory cytokines (IL-4) was reduced. Changes in cytokine pool probably reflect the differences in the severity of the pathological process in the muscle tissue of both genetic variations of mdx mice.

Cytokine profile of the blood in mice with normal and abnormal heart rhythm.

Differences in the pools of 10 cytokine were found in blood samples from the caudal vein of mice with normal and abnormal heart rhythm. Both groups were albino mice bred by us and differing from mdx albino mice by the absence of mutation in muscular dystrophin gene. Mice with normal heart rhythm had low IL-17 content and elevated concentrations of proinflammatory cytokines IL-6 and IL-1α in comparison with the normal (according to published data). In mice with bradyarrhythmias, increased blood levels of IL-10, IL-6, IL-5, IL-2, IL-1α, IL-17, IL-4, TNF-α, and granulocyte-macrophage colony-stimulating factor were detected. The relative content of IL-4 and IL-17 in the total cytokine pool increased. The lifespan of mice with bradyarrhythmias and cytokine hyperexpression was shorter by 2-3 months in comparison with mice without heart rhythm disturbances and moderate changes in the cytokine pool.

Intravenous xenotransplantation of human placental mesenchymal stem cells to rats: comparative analysis of homing in rat brain in two models of experimental ischemic stroke.

Mesenchymal stem cells from human placenta obtained after term natural delivery were cultured and labeled with vital dye Dil of magnetic fluorescing microparticles. The labeled cells were transplanted intravenously to rats with occlusion of the median cerebral artery. Penetration of cells through the brain-blood barrier and their distribution in the brain of experimental animals were studied on serial cryostat sections. Two models of cerebral artery occlusion associated with different traumatic consequences were used. The efficiency of crossing the blood-brain barrier by transplanted cells, the number of mesenchymal cells attaining the ischemic focus and neurogenic zones, and the time of death of transplanted cells largely depended on the degree and nature of injury to the central nervous system, which should be taken into account when planning the experiments for evaluation of the effects of cell therapy on the models of neurological diseases and in clinical studies in the field of regenerative neurology.

Detection of minor subpopulations of colorectal adenocarcinoma cells expressing cancer stem cell markers.

The expression of puitative surface molecular markers of cancer stem cells on human colorectal adenocarcinoma cells was analyzed by flow cytofluorometry. Cell subpopulations expressing markers of epithelial and malignant cells and stem cell markers were identified. Four minor subpopulations with CD24(+)/CD133(+), CD44(+)/CD133(+), CD90(+)/CD71(+), or CD90(+)/CD24(+) phenotypes meeting this requirement were detected; presumably, those were cancer stem cell subpopulations. These results extend our knowledge on heterogeneity of human colorectal adenocarcinoma cell population and outline new trends of research of cancer stem cell phenotype in these tumors.

Functional properties of mesenchymal stem cells labeled with magnetic microparticles in vitro and analysis of their distribution after transplantation.

Mesenchymal stem cells enzymatically isolated from human placenta were labeled with magnetic fluorescent microparticles (d=0.96 µ). We showed that microparticles in high doses (>10 µl stock suspension per 1 ml culture medium) significantly inhibited cell proliferation in culture. In our work we determined the optimal concentration of particles not affecting physiological properties of mesenchymal stem cells: it does not change cell proliferation, does not induce apoptosis, and does not modulate their transdifferentiation into neuronal cells. In vivo experiments showed that the chosen particles allow easy visualization of transplanted cells ex vivo on sections of different tissues.

A new genetic variant of mdx mice: study of the phenotype.

Genetic selection in a colony of mdx mice (suffering from X-chromosome-linked muscular dystrophy) resulted in generation of their new genetic variant. In this new variant, the genetic, biochemical, and histological markers of muscular dystrophy are combined with signs of oculocutaneous albinism (skin and fur depigmentation), transillumination of the iris, sharply reduced pigmentation of the retinal epithelium, and increase of the eyeball refraction). Two sensorimotor tests (negative geotaxis and wire back down hanging) detected other phenotypical characteristics of albino mdx mice carrying, in addition to the mutation in the dystrophin gene exon 23 (intrinsic of the "classical" black mdx mice), an extra mutation responsible for pigmentation disorders. Slow geotaxis, despite longer wire back down hanging capacity, was regarded as aggravation of the neurological dysfunction in albino mdx mice in comparison with black mdx mice.

Mechanisms of positive effects of transplantation of human placental mesenchymal stem cells on recovery of rats after experimental ischemic stroke.

Mesenchymal stem cells isolated from human placenta and in vitro labeled with fluorescent magnetic microparticles were intravenously injected to rats 2 days after induction of focal cerebral ischemia (endovascular model). According to MRT findings, transplantation of mesenchymal stem cells led to an appreciable reduction of the volume of ischemic focus in the brain. Two or three weeks after transplantation, labeled cells accumulated near and inside the ischemic focus, in the hippocampus, and in the subventricular zone of both hemispheres. Only few human mesenchymal stem cells populating the zone adjacent to the ischemic focus started expressing astroglial and neuronal markers. On the other hand, transplantation of mesenchymal stem cells stimulated proliferation of stem and progenitor cells in the subventricular zone and migration of these cells into the ischemic zone. Positive effects of transplantation of these cells to rats with experimental ischemic stroke are presumably explained by stimulation of proliferation of resident stem and progenitor cells of animal brain and their migration into the ischemic tissue and adjacent areas. Replacement of damaged rat neurons and glial cells by transplanted human cells, if it does take place, is quite negligible.

Development and introduction of production standards for cell products of mesenchymal origin.

The results of the development and introduction of universal production standards for cell products of mesenchymal origin are presented: technology for obtaining and culturing of primary cell cultures from human postnatal organs and tissues, cell product quality and safety control procedures, methods for cell product storage and transportation, and the necessary files.

Use of bone marrow mesenchymal (stromal) stem cells in experimental ischemic stroke in rats.

The effects of human mesenchymal stem cells on neurological functions and behavioral reactions of animals and on damaged brain tissue were studied on the model of focal cerebral ischemia in rats. Homing and differentiation of transplanted mesenchymal stem cells were also studied. Significant regression of neurological disorders after cell transplantation was noted, no appreciable shifts were detected by magnetic resonance tomography. Homing of transplanted cells was detected mainly in the zone of focal ischemia. Some cells died, others exhibited signs of differentiation into neurons and glia.

Capability of human mesenchymal cells isolated from different sources to differentiation into tissues of mesodermal origin.

We compared the capacity of cultured human skin fibroblasts, human umbilical cord cells obtained after normal delivery on gestation week 38-40, and mesenchymal bone marrow stem cells to differentiation into adipocytes, osteoblasts, and chondrocytes. Our findings suggest that mesenchymal stem cells are multipotent cells and can differentiate into adipose, cartilaginous, and bone tissue. Umbilical cord fibroblast-like cells can differentiate into adipocytes and chondrocytes, and only few cells in this culture can differentiate into osteoblasts. Skin fibroblasts differentiate only into adipocytes.

Comparatine analysis of cytophenotypes of cells of mesenchymal lineage isolated from human tissues.

The expression of cytoplasmic and surface proteins in cultured human skin fibroblasts, human umbilical cord cells obtained after normal delivery on gestation week 38-40, and mesenchymal bone marrow stem cells was compared by the methods of immunocytochemistry and flow cytofluorometry. Bone marrow mesenchymal stem cells expressed a great variety of marker proteins typical of stem and progenitor cells and did not express proteins typical of differentiated cells. Fibroblast-like umbilical cord cells expressed markers of both stem cells and differentiated cells. Fibroblasts of dermal origin were characterized by intensive expression of proteins typical of differentiated cells.

Regeneration of the rat spinal cord after thoracic segmentectomy: growth and restoration of nerve conductors.

The results presented in our previous report (Morfologiya, 127, No. 2 (2005)) provided evidence that consolidation of the spinal cord (SC) after thoracic segmentectomy in rats occurs as a result of the formation of a connective tissue scar, which is quicker when the defect is filled with collagen gel. The present report describes analysis of semithin sections and transmission electron microscopy studies demonstrating that regenerating nerve conductors traverse connective tissue in structures whose organization is identical to that of peripheral nerves. In the zone of SC rarefaction caudal to the trauma site, myelination of growing axons is mediated by glial cells without the formation of nerve trunks. Large numbers of fine regenerating conductors were seen at the sites of degenerated myelin fibers in the fasciculi of the white matter in the lumbar segments of the SC.

Modern cell technologies in the treatment of patients with tympanic membrane injury inflicted by mine explosion.

We evaluated the efficiency of transplantation of cultured human allofibroblasts onto tympanic membrane damaged by mine explosion in combination with Tampograss dressing (Paul Hartman). Transplantation of cultured human allofibroblasts was effective in 100% cases. Application of the film with fibroblasts onto perforation occupying from 1/4 to 1/2 of the tympanic membrane was more effective by 15% (by 59% in subtotal perforation) than tympanoplasty with amnion membrane. The mean duration of tympanic membrane restoration after spontaneous healing and amnionoplasty is virtually the same, while transplantation of allofibroblasts accelerated the process in comparison with other groups in perforation of any size; in subtotal defect the duration of tympanic membrane restoration was shorter by 14 +/- 1 days.

Mouse retinal progenitor cell (RPC) cocultivation with retinal pigment epithelial cell culture affects features of RPC differentiation.

We provide evidence that coculturing of retinal progenitor cells (RPC) with retinal pigment epithelial cells significantly biases the standard in vitro RPC differentiation patterns. In particular, in cocultivation experiments RPCs lost the ability to differentiate spontaneously and displayed approximately 2.1-2.4-fold increase in immunoreactivity to the neural stem cell marker nestin and approximately 1.6-1.7-fold increase in rod photoreceptor cell rhodopsin marker immunoreactivity. The data suggest the influence of the intercellular interaction networks on RPC differentiation.

Comparative study of adult human skin fibroblasts and umbilical fibroblast-like cells.

Expression of markers, collagens, and HLA-1 by human skin fibroblasts and fibroblast-like cells isolated from the umbilical Wharton's jelly was compared. Skin fibroblasts express collagens (proteins characteristic of differentiated cells of this histogenetic series) and HLA-1, while umbilical cells express, in addition to collagens, juvenile surface markers and almost no HLA-1. This indicates that fibroblast-like cells isolated from different sources are different and can serve as sources for the creation of cell preparations with different characteristics in future.

Regeneration of the rat spinal cord after thoracic segmentectomy: restoration of the anatomical integrity of the spinal cord.

Segmentectomy of the spinal cord (SC) was performed in 28 rats at the level of the tenth thoracic vertebra. Scar formation at the operation site was studied, along with the completeness of the anatomical integrity of the SC, in control animals (group 1) and after filling of the defect with the neural matrix Spherogel (group 2) and with Spherogel containing embryonic nerve cells (group 3). The defect in the SC in animals of the control group was filled with fibrin masses by 1-2 weeks, while connective tissues scars were already formed by this time in rats of experimental groups 2 and 3. By 10-11 weeks, these animals showed partial recovery of movement in the three lower limb joints. The scar tissue and adjacent zones of the SC showed large quantities of regenerating fine myelinated nerve fibers. These were clearly evident in the zones of the cranial and caudal margins, to the extent of the appearance of typical SC tissue. In preparations obtained from control animals, fine myelinated fibers were noted in the immediate vicinity of SC substance, while nerve fibers were few in number in scar tissue and the cellular bands of the transitional zone.

Cytofluorometric analysis of phenotypes of human bone marrow and umbilical fibroblast-like cells.

Comparative analysis of the expression of some surface markers of human bone marrow mesenchymal stem cells, umbilical fibroblast-like cells, and skin fibroblasts was carried out by the flow cytofluorometry method. Mesenchymal stem cells and umbilical fibroblast-like cells were similar by the levels of expression of the main histocompatibility complex antigens, adhesion molecules, and some growth factor receptors. The profile of skin fibroblast surface antigens was characterized by higher expression of the markers typical of differentiated cells. The results prove the possibility of using umbilical fibroblast-like cells as an alternative source of mesenchymal stem cells for cell replacement therapy.

Restoration of gastrocnemius muscle in MDX mice of different age after injury and implantation of xenogenic muscle tissue.

The intensity of regeneration of crossed gastrocnemius muscle was evaluated in two groups of mdx mice of different age 2 weeks after implantation of crushed muscle tissue from newborn rats into the wound defect area. The effect of xenoplasty manifested in increased weight of the damaged muscle. The effect was observed in mice aging 12-16 weeks but not in those aged 40-48-weeks. Structural changes in the skeletal muscle tissue intrinsic of mdx mice and augmenting with age were detected in intact mice before the experiment. Activity of muscle fiber regeneration in intact and injured muscle of 40-48-week-old mice was significantly lower than in 12-16-week-old ones. Myoblasts of the xenogenic transplant retained viability in recipient muscles for at least 2 weeks. posttraumatic regeneration was stimulated in only 12-16-week animals. Xenoplasty was ineffective in older animals and even somewhat enhanced the destructive processes in the muscle. It seems that age-specific regeneration activity of the recipient skeletal muscle tissue should be taken into consideration in the development of effective strategy of cell therapy for progressive muscular dystrophy.

Cardiotropic effect of extracardiac transplantation of embryonic human myoblasts to mice with bradycardia: various effects of cell material.

Animals with bradycardia were detected in reproductive colony of mdx mice. Low pulse rate was associated with poor survival and predisposition to sudden death, but did not directly depend on the presence of dystrophin mutant gene or animal age. Heart rate increased in old mice with bradycardia after extracardial, intramuscular, and intravenous injection of human embryonic myoblasts. Stable normalization of the pulse was observed 2 weeks after transplantation, but early peak of heart rate was observed as early as 24 h after cell transplantation. Cell suspensions, which could contain stem cells (blood mononuclears and CD34+ lymphocytes), also corrected heart rhythm. Unlike the effect of myoblasts, cardiotropic effect of mononuclears was preceded by a period of tachycardia, while the effect of CD34+ lymphocytes was very unstable. The cardiotropic effect of myoblasts was combined with life span prolongation and certain rejuvenation in some animals. Erythrocytes and supernatant obtained during blood cell fractionation did not modify the heart rhythm in mice with bradycardia. After injection of myoblasts to mice with rare and normal pulses serum creatine kinase activity decreased with different rates. These data attest to a variety of biological effects of stem cells and/or their derivatives and to ambiguous mechanisms of these effects.