Comparative Analysis of the Different Dyes' Potential to Assess Human Normal and Cancer Cell Viability In Vitro under Different D/H Ratios in a Culture Medium.

In this study, using new approach (laser diffraction + biological dyes), we have demonstrated the decrease of cells viability in vitro in the deuterated growth medium, whereas in the deuterium-depleted medium, there was an increase of cell viability. We have also found that not all dyes are equally sensitive to the D/H ratios in the culture medium (system) as well as to the different cell types (cancer vs normal cells).

Postnatal extra-embryonic tissues as a source of multiple cell types for regenerative medicine applications.

AIM: We aimed to isolate and characterize the cell types which could be obtained from postnatal extra-embryonic tissues. MATERIALS AND METHODS: Fresh tissues (no more than 12 h after delivery) were used for enzymatic or explants methods of cell isolation. Obtained cultures were further maintained at 5% oxygen. At P3 cell phenotype was assessed by fluorescence-activated cell sorting, population doubling time was calculated and the multilineage differentiation assay was performed. RESULTS: We have isolated multiple cell types from postnatal tissues. Namely, placental mesenchymal stromal cells from placenta chorionic disc, chorionic membrane mesenchymal stromal cells (ChM-MSC) from free chorionic membrane, umbilical cord MSC (UC-MSC) from whole umbilical cord, human umbilical vein endothelial cells (HUVEC) from umbilical vein, amniotic epithelial cells (AEC) and amniotic MSC (AMSC) from amniotic membrane. All isolated cell types displayed high proliferation rate together with the typical MSC phenotype: CD73+CD90+CD105+CD146+CD166+CD34-CD45-HLA-DR-. HUVEC constitutively expressed key markers CD31 and CD309. Most MSC and AEC were capable of osteogenic and adipogenic differentiation. CONCLUSION: We have shown that a wide variety of cell types can be easily isolated from extra-embryonic tissues and expanded ex vivo for regenerative medicine applications. These cells possess typical MSC properties and can be considered an alternative for adult MSC obtained from bone marrow or fat, especially for allogeneic use.

Tissue-engineered bone for treatment of combat related limb injuries.

AIM: Based on our preliminary positive clinical results with use of cultured bone marrow-derived multipotent mesenchymal stem/stromal cells in traumatology, our aim was to develop living three-dimensional tissue-engineered bone equivalent transplantation technology for restoration of critical sized bone defects caused by combat related high energy trauma. MATERIALS AND METHODS: To fabricate bone equivalent we used devitalized allogeneic bone scaffolds (blocks and chips) seeded with cultured autologous cells: bone marrow-derived multipotent mesenchymal stem/stromal cells in mix with periosteal progenitor cells and endothelial progenitor cells. Quality/identity of cell cultures was assured by donor and cell culture infection screening (immunofluorescence assay, polymerase chain reaction), flow cytometry (cell phenotype), karyotyping (GTG banding), functional assays (colony forming units analysis, multilineage differentiation assay). Bone defect treatment with bone equivalent application was fully completed in 39 combat-injured with 42 defects. New bone formation was assessed by the radiographic examination. RESULTS: Casualties were included in a treatment program an average of 10.1 months after injury, provided the ineffectiveness of conventional surgery methods. All cell type cultures had a normal karyotype and appropriate phenotype, differentiation potential and functional properties, ~30% colony forming units frequency and hadn't any signs of cell senescence. The fluorescein diacetate/propidium iodide combined staining and histology analysis of graft samples before transplantation showed their regular seeding with viable cells. Pathomorphological analysis of bone equivalent specimens 3-6 months post-op revealed the active remodeling processes and immature bone tissue formation. Bone defect restoration was observed 5-6 months post-op. CONCLUSION: The developed biotechnology of living three-dimensional tissue-engineered bone equivalent transplantation with overall effectiveness 90.4% allows restoring the bone integrity, forming new bone tissue in a site of bone defect, and significantly reducing the rehabilitation period of a patient.

Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications.

AIM: The purpose of this work was to obtain, multiply and characterize the adult neural crest-derived multipotent stem cells from human hair follicle for their further clinical use. MATERIALS AND METHODS: Adult neural crest-derived multipotent stem cells were obtained from human hair follicle by explant method and were expanded at large-scale up to a clinically significant number. The resulted cell cultures were examined by flow cytometry and immunocytochemical analysis. Their clonogenic potential, ability to self-renewal and directed multilineage differentiation were also investigated. RESULTS: Cell cultures were obtained from explants of adult human hair follicles. Resulted cells according to morphological, phenotypic and functional criteria satisfied the definition of neural crest-derived multipotent stem cells. They had the phenotype Sox2+Sox10+Nestin+CD73+CD90+CD105+CD140a+CD140b+CD146+CD166+CD271+CD349+ CD34-CD45-CD56-HLA-DR-, showed high clonogenic potential, ability to self-renewal and directed differentiation into the main derivatives of the neural crest: neurons, Schwann cells, adipocytes and osteoblasts. CONCLUSION: The possibility of a large-scale expansion of adult neural crest-derived multipotent stem cells up to 40-200·106 cells from minimal number of hair follicles with retention of their phenotype and functional properties are the significant step towards their translation into the clinical practice.Key Words: regenerative medicine, neural crest, hair follicle, neural crest-derived multipotent stem cells, directed differentiation, large-scale expansion.

Endometrial stromal cells: isolation, expansion, morphological and functional properties.

AIM: We aimed to study biological properties of human endometrial stromal cells in vitro. MATERIALS AND METHODS: The endometrium samples (n = 5) were obtained by biopsy at the first phase of the menstrual cycle from women with endometrial hypoplasia. In all cases, a voluntary written informed consent was obtained from the patients. Endometrial fragments were dissociated by enzymatic treatment. The cells were cultured in DMEM/F12 supplemented with 10% FBS, 2 mМ L-glutamine and 1 ng/ml FGF-2 in a multi-gas incubator at 5% CO2 and 5% O2. At P3 the cells were subjected to immunophenotyping, multilineage differentiation, karyotype stability and colony forming efficiency. The cell secretome was assessed by BioRad Multiplex immunoassay kit. RESULTS: Primary population of endometrial cells was heterogeneous and contained cells with fibroblast-like and epithelial-like morphology, but at P3 the majority of cell population had fibroblast-like morphology. The cells possessed typical for MSCs phenotype CD90+CD105+CD73+CD34-CD45-HLA-DR-. The cells also expressed CD140a, CD140b, CD146, and CD166 antigents; and were negative for CD106, CD184, CD271, and CD325. Cell doubling time was 29.6 ± 1.3 h. The cells were capable of directed osteogenic, adipogenic and chondrogenic differentiation. The cells showed 35.7% colony forming efficiency and a tendency to 3D spheroid formation. The GTG-banding assay confirmed the stability of eMSC karyotype during long-term culturing (up to P8). After 48 h incubation period in serum-free medium eMSC secreted anti-inflammatory IL-1ra, as well as IL-6, IL-8 and IFNγ, angiogenic factors VEGF, GM-CSF and FGF-2, chemokines IP-10 and MCP-1. CONCLUSION: Thus, cultured endometrial stromal cells meet minimal ISCT criteria for MSC. Proliferative potential, karyotype stability, multilineage plasticity and secretome profile make eMSC an attractive object for the regenerative medicine use.