Effect of Storage Conditions on the Integrity of Human Umbilical Cord Mesenchymal Stromal Cell-Derived Microvesicles.

We studied the effect of storage conditions on the safety of microvesicles produced by human multipotent umbilical cord mesenchymal stromal cells into the conditioned medium. It was found that microvesicles can be stored without serious degradation for up to 1 week at 4°Ð¡, but were almost completely destroyed during freezing and thawing cycles irrespective of the storage temperatures (-20°Ð¡, -70°Ð¡, or -196°Ð¡). Similar results were obtained for lyophilized medium conditioned by human multipotent umbilical cord mesenchymal stromal cells. Addition of a cryoprotectant (5-10% DMSO) followed by freezing and/or lyophilization preserved microvesicles at a nearly initial level. These findings indicate that during storage, microvesicles, being membrane structures, behave similar to living cells and require appropriate conditions for prolonged storage.

Comparative Analysis of Secretome of Human Umbilical Cord- and Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells.

Production of cytokines and growth factors by cultured human umbilical cord tissue- and bone marrow-derived multipotent mesenchymal stromal cells was measured by multiplex analysis. In most cases, the concentrations of bioactive factors in the culture medium conditioned by umbilical cord-derived cells was ten- to hundred-times higher than in the medium conditioned by bone marrow-derived cells. These results suggest that both multipotent mesenchymal stromal cells from the umbilical cord and cell-free products can have more pronounced therapeutic effect in comparison with mesenchymal stromal cells obtained from "adult" sources.

Human Umbilical Cord Mesenchymal Stromal Cell-Derived Microvesicles Express Surface Markers Identical to the Phenotype of Parental Cells.

Production of microvesicles in culture of human umbilical cord multipotent mesenchymal stromal cells was studied and comparative analysis of the expression of some surface molecules (clusters of differentiation, CD) was performed. It was found that the mesenchymal stromal cells produce microvesicles in the amount sufficient for their detection by flow cytometry. Parallel analysis of the phenotypes of maternal mesenchymal stromal cells and secreted microvesicles revealed identical expression of surface molecules CD13, CD29, CD44, CD54, CD71, CD73, CD90, CD105, CD106, and HLA-I. The concentration of microvesicles in the conditioned medium was 17.9±4.6×106/ml; i.e. one cell produced ~40-50 (44.7±11.5) microvesicles over 2 days in culture.

Human Umbilical Cord Mesenchymal Stromal Cells Support Viability of Umbilical Cord Blood Hematopoietic Stem Cells but not the "Stemness" of Their Progeny in Co-Culture.

Cell-cell interactions and the ability of mesenchymal stromal cells to support the expansion of hematopoietic progenitor cells were studied in co-culture of human umbilical cord tissue-derived mesenchymal stromal cells and nucleated umbilical cord blood cells. It was found that hematopoietic stem cells from the umbilical cord blood are capable to adhere to mesenchymal stromal cells and proliferate during 3-4 weeks in co-culture. However, despite the formation of hematopoietic foci and accumulation of CD34+ and CD133+ cells in the adherent cell fraction, the ability of newly generated blood cells to form colonies in semi-solid culture medium was appreciably reduced. These findings suggest that human umbilical cord tissue-derived mesenchymal stromal cells display a weak capability to support the "stemness" of hematopoietic stem cell progeny despite long-term maintenance of their viability and proliferation.

Human Umbilical Cord Blood Serum: Effective Substitute of Fetal Bovine Serum for Culturing of Human Multipotent Mesenchymal Stromal Cells.

Optimal conditions for culturing of multipotent mesenchymal stromal cells in the presence of pooled umbilical cord blood serum were determined. It was found that umbilical cord blood serum in a concentration range of 1-10% effectively supported high viability and proliferative activity of cells with unaltered phenotype and preserved multilineage differentiation capacity. The proposed approach allows avoiding the use of xenogenic animal sera for culturing of multipotent mesenchymal stromal cells and creates prerequisites for designing and manufacturing safe cellular and/or acellular products for medical purposes.

Expression of Surface Molecules in Human Mesenchymal Stromal Cells Co-Cultured with Nucleated Umbilical Cord Blood Cells.

We studied the expression of different classes of surface molecules (CD13, CD29, CD40, CD44, CD54, CD71, CD73, CD80, CD86, CD90, CD105, CD106, CD146, HLA-I, and HLA-DR) in mesenchymal stromal cells from human umbilical cord and bone marrow during co-culturing with nucleated umbilical cord blood cells. Expression of the majority of surface markers in both types of mesenchymal stromal cells was stable and did not depend on the presence of the blood cells. Significant differences were found only for cell adhesion molecules CD54 (ICAM-1) and CD106 (VCAM-1) responsible for direct cell-cell contacts with leukocytes and only for bone marrow derived cells.

Isolation of Multipotent Mesenchymal Stromal Cells from Cryopreserved Human Umbilical Cord Tissue.

Umbilical cord stroma is an easily available, convenient, and promising source of multipotent mesenchymal stromal cells for regenerative medicine. Cryogenic storage of umbilical cord tissue provides more possibilities for further isolation of multipotent mesenchymal stromal cells for autologous transplantation or scientific purposes. Here we developed a protocol for preparation of the whole umbilical cord tissue for cryogenic storage that in combination with the previously described modified method of isolation of multipotent mesenchymal stromal cells allowed us to isolate cells with high proliferative potential, typical phenotype, and preserved differentiation potencies.

Changes in Cell Composition of Umbilical Cord Blood and Functional Activity of Hematopoietic Stem Cells during Cryogenic Storage and Repeated Freezing/Thawing Cycles.

We analyzed changes in cell composition of umbilical cord blood and functional activity of hematopoietic stem cells during cryogenic storage and after repeated freezing/thawing cycles. It was found that repeated freezing/thawing cycles performed according to the optimal programmable freezing protocol did not significantly affect viability and functional activity of hematopoietic stem cells. When fast freezing program was used, the cells completely lost their capacity to form colonies in semisolid medium, despite high viability parameters in the test with 7-AAD.

Optimized Protocol for Isolation of Multipotent Mesenchymal Stromal Cells from Human Umbilical Cord.

Extraembryonic tissues, in particular, umbilical cord stroma are promising sources of multipotent mesenchymal stromal cells for regenerative medicine. In recent years, methods for isolation of mesenchymal stromal cells from different compartments of the umbilical cords based on enzymatic disaggregation of the tissue or on tissue explants have been proposed. Here we propose a protocol of isolation of multipotent mesenchymal stromal cells from the whole umbilical cord that combines the advantages of each approach and ensures sufficient cell yield for further experimental and clinical applications. A combination of short-term incubation of tissue fragments on cold collagenase solution followed by their culturing in the form of explants significantly increased the yield of cells with high proliferative activity, typical pluripotent mesenchymal stromal cell phenotype, and preserved differentiation capacity.

Umbilical cord blood for autologous transfusion in the early postnatal ontogeny: analysis of cell composition and viability during long-term culturing.

Changes in cell composition and viability as well as the content and functional activity of hemopoietic progenitor cells were analyzed during long-term (up to 1 month at 4°C) storage of human umbilical cord blood cells. No significant quantitative changes in erythrocytes were found during this period. The total content and viability of leukocytes changed, which resulted in the prevalence of mononuclear cells (lymphocytes and monocytes). Analysis of functional activity of hemopoietic stem cells in semisolid culture revealed a decrease in the relative content of CFU during the first week of storage [corrected] and inability of cells to colony formation after 2 weeks.

Enrichment of umbilical cord blood mononuclears with hemopoietic precursors in co-culture with mesenchymal stromal cells from human adipose tissue.

We demonstrated the possibility of enrichment of umbilical cord blood mononuclear fraction with early non-differentiated precursors under conditions of co-culturing with mesenchymal stromal cells from the human adipose tissue. It was established that umbilical cord blood mononuclear cells adhered to mesenchymal stromal cell feeder and then proliferate and differentiate into hemopoietic cells. In comparison with the initial umbilical cord blood mononuclear fraction, the cell population obtained after 7-day expansion contained 2-fold more CFU and 33.4 ± 9.5 and 24.2 ± 11.2% CD34(+) and CD133(+) cells, respectively, which corresponds to enrichment of precursor cell population by 148 ± 60. The proposed scheme of expansion of hemopoietic cells from umbilical cord blood is economically expedient and can widely used in biology and medicine.

Specific interaction of cultured human mesenchymal and hemopoietic stem cells under conditions of reduced oxygen content.

We studied the effect of reduced oxygen content (5%) on the phenotype and functional activity of cultured human mesenchymal stem cells. The expression of main immunophenotypic markers for mesenchymal stem cells (CD13, CD29, CD44, CD73, CD90, CD105, and HLA-I) remained practically unchanged under conditions of hypoxia. The expression of cell adhesion molecules (CD54 and CD106) increased during coculturing of mesenchymal stem cells and hemopoietic stem cells. These changes were accompanied by increased production of hemopoietins (interleukin-6 and interleukin-8) and enhanced colony-forming capacity of hemopoietic stem cells. Coculturing of mesenchymal stem cells and hemopoietic stem cells during hypoxia was followed by increased formation of hemopoietic islets and intensive production of interleukin-6, interleukin-8, and vascular endothelial growth factor (compared to cultures under normoxic conditions).

Cell phenotypes in human amniotic fluid.

Stem cells capable of long-term proliferation and differentiation into different cell types may be a promising source of cells for regenerative medicine. Recently, much attention has been paid to fetal stem cells, among which are cells from amniotic fluid (AF). We have isolated amniotic stem cells from 3 AF samples. Flow cytometry, RT -PCR and immunohistochemistry have shown that these cells express mesenchymal (CD90, CD73, CD105, CD13, CD29, CD44, and CD146), neural (≤3-tubulin, Nestin, and Pax6), epithelial (keratin 19 and p63) markers and also markers of pluripotency (Oct4, Nanog, and Rex-1). Transplantation of the cells to nude mice does not lead to tumor formation. Thus, putative stem/progenitor cells from AF are capable of long-term proliferation in vitro and the profile of gene expression led us to speculate that they have greater differentiation potential than mesenchymal stem cells and may be useful for cell therapy.

Optimum conditions for culturing of human bone marrow and adipose tissue mesenchymal precursor cells.

We present a technology of culturing of human mesenchymal stem cells under conditions excluding the presence of animal sera or additional growth factors, but preserving high proliferative potential and the capacity of these cells to multilineage differentiation. Human umbilical serum was used as the alternative material. We found that in the presence of human umbilical serum mesenchymal stem cells more effectively proliferate and retain their differentiation capacity. The proposed technology yields 109-1010 morphologically and functionally identical cells.

[Alterations in actin cytoskeleton and rate of reparation of human endothelium (the wound-healing model) under the condition of clinostatting]. / Izmeneniia aktinovogo tsitoskeleta i skorosti reparatsii mekhanicheski povrezhdennogo monosloia éndoteliia cheloveka v usloviiakh klinostatirovaniia.

Effects of long-term simulation of hypogravity on actin cytoskeleton and cell migration were investigated in cultured human endothelium cells (EC). In control, F-actin resided predominantly on the periphery of cell forming an array of parallel bundles with "dense bodies" along the edge. A small number of actin cable fibers was found in the center. Already after 1-2 hrs of clinostatting at 5 RPM the cell cytoskeleton showed actin filament thinning and displacement toward the cell edges. In subsequent 6-18 hrs, almost all actin fibers had left the center part of EC and had ranged themselves in a continuous F-actin line in the intercellular contact area. In most cases, these changes resulted in the so-called "ruff-edge". Since both the disappearance of cable fibers and formation of the "ruff-edge" add to the cell migration activity, this parameter was studied with the would-healing model. According to our data, 24-48 hrs of exposure to hypogravity stimulates cell migration and expedites 2-3 times reparation of mechanically damaged monolayer. The results suggest that effects of hypogravity on cultured human EC are likely to be consequent to alterations in the activity of protein kinase C and/or adenylate cyclase involving many members of the cellular metabolism.

Expression of cell adhesion molecules and lymphocyte-endothelium interaction under simulated hypogravity in vitro.

Using histochemical staining and FACS-analysis we have studied the basal and TNF-alpha induced expression of E-selectin, ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (ECs) exposed to simulated hypogravity. Control ECs did not contain detectable amounts of E-selectin or VCAM-1 but were ICAM-1 positive. As soon as after 6-8 hrs of clinorotation at 5 RPM the cellular content of ICAM- 1 increased. Moreover, hypogravity potentiated the effect of inflammatory cytokines (TNF-alpha and IL-1) on ICAM-1 expression. No increase in E-selectin or VCAM-1 expression was observed in ECs exposed to hypogravity itself. However, hypogravity reduced E-selectin and VCAM-1 expression in cell cultures activated by cytokines, more visible at their low (5-10 U/ml) concentrations. Both, control and clinorotated ECs poorly supported spontaneous lymphocyte adhesion; the adhesion of PMA-activated leukocytes was 15-20-fold higher. The interaction of unstimulated lymphocytes with cytokine-activated endothelium was more noticeable but significantly lower in cultures exposed to hypogravity. Activated blood cells interacted with endothelium more effectively, particularly, under hypogravity. Obtained results suggest that EC adhesion molecule expression and endothelium-lymphocyte interaction are altered under simulated hypogravity conditions in direction of increase of endotlielial adhesiveness for activated blood cells.

[Morphological and functional study of human endothelium response to hyperthermia in vitro]. / Morfologicheskie i funktsional'nye osobennosti otveta éndoteliia cheloveka na gipertermiiu in vitro.

Effects of hyperthermia (+42.5 degrees C) on the endothelial monolayer organization, actin cytoskeleton and cell viability have been examined in culture of human aortic endothelial cells (EC). Short-lasting hyperthermia (1-4 hrs) provoked disappearance of stress fibers, redistribution of actin filaments to the area of cell-to-cell contacts, shape changes and reorganization of the monolayer. It also stimulated formation of intercellular contacts in a preconfluent EC culture. The cAMP content was elevated one hour after heat treatment and then lowered to negligible values (comparing to the basal cAMP level). Long-lasting hyperthermia (6-72 hrs) resulted in EC injury, and damage of endothelial monolayer accompanied by increased Chromium-51 release and almost complete blockade of [3H]-thymidine incorporation into DNA. Addition of cAMP elevating drugs (forskolin, 8-Br-cAMP or isobutylmethylxanthine) into the cell culture medium prevented heat-induced decrease in cAMP concentration, stimulated EC spreading, protected EC from injury, and promoted integrity of endothelial monolayers. Obtained results indicate that long-lasting hyperthermia can be regarded as an additional factor of endothelium injury involved in development of vascular pathology.

[Gravitation sensitivity of human endothelium]. / Gravichuvstvitel'nost' éndoteliia cheloveka.

Experiments on the effects of hypogravity (clinostatting) on growth and formation of monolayer of cultivated endothelium cells of the human umbilical vein demonstrated sensitivity of endothelium to the gravitational stimulus as it responded by a significant reduction of the proliferative activity of cells in culture. The most favorable conditions for cultivating endothelium cells under extended (15-d) hypogravity in vitro were determined.

[Coculture of human endothelium and smooth muscle cells: functional heterogeneity of endothelial cells from zones with different susceptibility to atherosclerosis]. / Sovmestnoe kul'tivirovanie éndotelial'nykh i gladkomyshechnykh kletok aorty cheloveka: funktsional'naia geterogennost' éndoteliia v zonakh s razlichnoi predraspolozhennost'iu k aterosklerozu.

Using co-culture technique and 3H-thymidine radioautography we have studied effects of human aortic endothelial cells (EC), isolated separately from zones of low (LP) and high (HP) probability of atherosclerosis of grossly normal and atherosclerotic aortas, on 3H-thymidine incorporation by human intimal smooth muscle cells (SMCs). It was found that EC activity depended on a zone of probability, from which the cells were isolated, and on the degree of atherosclerotic lesion. The first-passage ECs from grossly normal LP zones inhibited 3H-thymidine incorporation, compared to control, incubated without Ecs, SMCs (63.5 +/- 27.5%). Cells from HP zones of the same vessels were less active or stimulated SMC proliferation (99.4 +/- 42.9%). EC cultures obtained from both LP and HP zones of atherosclerotic vessels had, as a rule, no effect or increased 3H-thymidine incorporation by SMCs (100.3 +/- 19.8 and 124.1 +/- 20.1%). In contrast to morphologically heterogeneous primary and first-passage cultures obtained from high seeding density, EC monolayers obtained with a split 1:10 were composed predominantly of small mononuclear cells. These cultures effectively inhibited SMC DNA synthesis independently of a zone of probability and a degree of atherosclerotic lesion of aorta (60.4 +/- 10.0 and 51.5 +/- 12.7%). The obtained data suggest that EC morphological heterogeneity is accompanied by functional changes of cells and may be involved in atherosclerotic plaque formation.