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Aim. Neural progenitor cells (NPC) are used for the development of cell therapies of neurological diseases. Their stereotaxic transplantation in the middle cerebral artery occlusion (MCAO) model imitating ischemic stroke results in symptom aleviation. However, exploration of less invasive transplantation options is essential, because stereotaxic transplantation is a complex procedure and can be applied to humans only by vital indications in a specialized neurological ward. The aim of the present study was to evaluate the efficacy of cell therapy of the experimental ischemic stroke by the intra-arterial transplantation of NPC.Materials and methods. NPC for transplantation (IPSC-NPC) were derived by two-stage differentiation of cells of a stable line of human induced pluripotent stem cells. Stroke modeling in rats was carried out by transitory 90 min endovascular MCAO by a silicon-tipped filament. NPC were transplanted 24 hours after MCAO. Repetitive magnetic resonance tomography of experimental animals was made with the Bruker BioSpin ClinScan tomograph with 7 Tl magnetic field induction. Animal survival rate and neurological deficit (using mNSS standard stroke severity scale) were evaluated at the 1st (before IPSC-NPC transplantation), 7th and 14th day after transplantation. Histological studies were carried out following standard protocols.Results. Intra-arterial transplantation of 7 × 105 IPSC-NPC in 1 ml at a constant 100 l/min rate in case of secured blood flow through the internal carotid artery did not cause brain capillary embolism, additional cytotoxic brain tissue edemas or other complications, while inducing increase of animal survival rate and enhanced revert of the neurological deficit. IPSC-NPC accumulation in brain after intra-arterial infusion was demonstrated. Some cells interacted with the capillary endothelium and probably penetrated through the blood-brain barrier.Conclusion. Therapeutic efficacy of the systemic, intra-arterial administration of NPC in ischemic stroke has been experimentally proven. A method of secure intra-arterial infusion of cell material into the internal carotid artery middle in rats has been developed and tested.

Human placenta mesenchymal stromal cells were injected to healthy rats either stereotaxically into the striatum or intra-arterially through the internal carotid artery. Some cells injected into the brain migrated along the corpus callosum both medially and laterally or concentrated around small blood vessels. A small fraction of MSC injected intra-arterially adhered to the endothelium and stayed inside blood vessels for up to 48 hours mostly in the basin of the middle cerebral artery. Neither stereotaxic, nor intra-arterial transplantation of mesenchymal stromal cells modulated the proliferation of neural stem cells in the subventricular zone of the brain, but stereotaxic transplantation suppressed activation of their proliferation in response to traumatization with the needle.

The cells isolated from biopsy specimen of a patient with alcoholic liver cirrhosis and cultured under standard conditions for obtaining stromal cell culture clearly diverged during early passages into two morphologically and phenotypically different subtypes: epithelial and mesenchymal. Mesenchymal cells expressed CD90 and CD44 and epithelial cells expressed CD166, CD227, and hepatocyte growth factor receptor Met. Starting from passage 6, the culture underwent spontaneous morphological changes and by passages 8-10 contained only epithelium-like cells. CD90 and CD44 expression disappeared, CD166 and CD227 expression remained unchanged, and Met expression increased. A small fraction of cells expressed GATA-4, HNF3β, HNF1α, and HNF4α. After addition of inducers of hepatogeneic differentiation, the cells started producing albumin.

We compared the effects of placental mesenchymal stromal cells and neural progenitor cells derived from induced human pluripotent cells after their intravenous administration to rats in 24 h after transitory occlusion of the middle cerebral artery. The therapeutic effects were evaluated by the dynamics of animal survival, body weight, neurological deficit, and the volume of infarction focus in 7, 14, 30, and 60 days after surgery. Intravenous injection of neural progenitor cells produced a therapeutic effect on the course of experimental ischemic stroke by increasing animal survival in the most acute period and accelerating compensation of neurological deficit and body weight recovery. Neural progenitor cells were more effective than mesenchymal stromal cells from human placenta. The effectiveness of intravenous transplantation of neural progenitor cells in the model of occlusion of the middle cerebral artery is shown by us for the first time, although the therapeutic effect of their direct transplantation into the brain has already been described.

It is demonstrated that the output optical signal of MTT test is directly proportional to the number of viable cells in the primary culture of mesenchymal cells (skin fi broblasts and mesenchymal stem cells from the bone marrow, placenta, and umbilical cord). The slope of the best curve in coordinates “cell number – optical signal” refl ecting specifi c productivity of MTT-formazan characterizes mean dehydrogenase activity of cells and their physiological activity. It was found that in vitro dehydrogenase activity of primary cultures of mesenchymal cells increased during the fi rst 3-5 passages and then tended to decrease. The variant of MTT method presented here can be used for standardization of cell materials.

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.

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.

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.

We studied immunoregulatory properties of cultured human stem cells of mesenchymal and ectodermal origins after their administration to mice. Xenotransplantation of mesenchymal stem cells from human placenta reduced the number of CD11c(+)dendritic cells in mouse spleens, but did not affect activation of dendritic cells from mouse spleen in culture. It was also shown that splenocytes isolated from animals 10 days after transplantation of mesenchymal stem cells more actively proliferated in response to the polyclonal stimulation. At the same time, transplantation of neither mesenchymal nor neural stem cells affected the ratio of CD4(+)/CD8(+)T cells and their total content in the peripheral blood in comparison with the corresponding parameters in the control groups.

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.

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.

Cultures of mesenchymal cells from human deciduous tooth pulp were derived. The phenotype and capacity to osteogenic and adipogenic differentiation of these cells are close to those of bone marrow mesenchymal stem cells. Deciduous tooth pulp mesenchymal cells populate biodegradable polylactide scaffolds and hence, can be used for the creation of tissue engineering transplants for bone defect repair. Storage of decidual tooth pulp mesenchymal cells in the stem cell cryobanks together with umbilical blood will appreciably extent the periods of age for collection of juvenile autologous stem cells for use throughout the life span.

Cell-mediated cytotoxicity was studied in vitro during the interaction of bone marrow mesenchymal stem cells, fibroblast-like cells from newborn umbilical cord, and skin fibroblasts of an adult donor with peripheral blood mononuclear cells. Independently on the origin, mesenchymal cells were not lysed with allogeneic natural killer cells and cytotoxic lymphocytes. Mixed cultures of mesenchymal cells had no cytotoxic effect on peripheral blood mononuclear cells and did not activate proliferation of T and B lymphocytes, natural killer cells, and CD14+ lymphocytes. In vitro experiments showed that mesenchymal cells of different origin and allogeneic immunocompetent cells are tolerant to each other.

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.

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.

An enzyme immunoassay is proposed for detection of antibodies in complexes with antigens. Specific anti-HIV antibodies were detected in sera which were considered HIV-negative.

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.

The preclinical analysis of the effectiveness of AZT and new compounds (sulphatized derivate of chitosan (Sch) and adamantileted Sch, contents 10 and 18% of adamantine) prepared on the basis of chitosan (Ch), carried out with the use of the experimental clinical test system (the in vitro model of peripheral blood mononuclears of a concrete patient) developed in laboratory, is presented. Both combinations in concentration 0.01 mkg/ml are decreased HIV antigens till 6.4 and 8.6% accordingly, while whilst initial Sch hare caused less effect in 4-5 fold.